Methods and systems for integrated on-chip single-molecule detection

ABSTRACT

The present disclosure provides methods and systems for performing single-molecule detection using fabricated integrated on-chip devices. In an aspect, the present disclosure provides a method for on-chip detection of an array of biological, chemical, or physical entities, comprising: (a) providing an array of light sensing devices; (b) immobilizing the array of biological, chemical, or physical entities on a substrate of the array of light sensing devices; (c) exposing the array of biological, chemical, or physical entities to electromagnetic radiation sufficient to excite the array of biological, chemical, or physical entities, thereby producing an emission signal of the array of biological, chemical, or physical entities; (d) using the array of light sensing devices, acquiring pixel information of the emission signal of the array of biological, chemical, or physical entities without scanning the array of light sensing devices across the array of biological, chemical, or physical entities; and (d) detecting the array of biological, chemical, or physical entities based at least in part on the acquired pixel information.

CROSS-REFERENCE

This application is a continuation application of U.S. application Ser.No. 17/513,877, filed Oct. 28, 2021, which is a continuation applicationof International Patent Application No. PCT/US2020/030501, filed Apr.29, 2020, which claims the benefit of U.S. Provisional Application No.62/840,209, filed Apr. 29, 2019, each of which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Biological assays may be used for applications such as genome sequencingor protein expression. It may be beneficial to tailor the design ofbiological assays for the fast, high-confidence identification of alarge number of small amounts of different biological materials.However, such requirements may introduce challenges in the form ofcompeting constraints on the design and fabrication of chips, flowcells, and detection systems used for such assays. For example, thelarge number of objects to be detected may impose constraints on theamount of material that can be used for each object and on the densityat which these objects can be loaded on a substrate of reasonable size.These limitations in turn may imply that only limited amount of signalis available for detection and that the signal of neighboring objectsbecomes difficult to differentiate. Further, the limited signal amountemitted by each object can negatively impact a detection time needed todetect the objects.

SUMMARY OF THE INVENTION

The present disclosure provides methods and systems for performingsingle-molecule detection using fabricated integrated on-chip devices.Using disclosed methods and systems, single-molecule detection can beperformed while achieving advantages such as: a reduction in thescanning time required by using a large number of light sensors forparallel imaging without moving parts during imaging, a reduction innoise levels by reducing the number of components in the imaging system,an improved resolution arising from detecting one object on each sensor,decreased crosstalk between neighboring object signals, and improveddetection sensitivity arising from improved light collection enabled bymicroscale or nanoscale features on the imaging sensors.

In an aspect, the present disclosure provides a method for on-chipdetection of an array of biological, chemical, or physical entities,comprising: (a) providing an array of light sensing devices; (b)immobilizing the array of biological, chemical, or physical entities ona substrate of the array of light sensing devices; (c) exposing thearray of biological, chemical, or physical entities to electromagneticradiation sufficient to excite the array of biological, chemical, orphysical entities, thereby producing an emission signal of the array ofbiological, chemical, or physical entities; (d) using the array of lightsensing devices, acquiring pixel information of the emission signal ofthe array of biological, chemical, or physical entities without scanningthe array of light sensing devices across the array of biological,chemical, or physical entities; and (e) detecting the array ofbiological, chemical, or physical entities based at least in part on theacquired pixel information.

In some embodiments, the electromagnetic radiation sufficient to excitethe array of biological, chemical, or physical entities comprises one ormore wavelengths of light. In some embodiments, the array of biological,chemical, or physical entities comprises biological, chemical, orphysical entities selected from the group consisting of: (i) a singlestructured nucleic acid particle (SNAP); (ii) a single SNAP with atleast one fluorescent label; (iii) a DNA origami; (iv) a DNA origamiwith at least one fluorescent label; (v) a single protein (antibody,antigen, peptide, aptamer, or other proteins); (vi) a single protein(antibody, antigen, peptide, aptamer, or other proteins) bound to asingle SNAP; (vii) a single protein (antibody, antigen, peptide,aptamer, or other proteins) bound to a single DNA origami, one or morefluorescent probes bound to a biological, chemical, or physical entityof (i)-(vii); (ix) one or more nanoparticles (e.g., organic, inorganic,or biological); (x) one or more nanoparticles with optical properties(e.g., quantum dots); (xi) one or more formulations of dendrimers; and(xii) a combination thereof.

In some embodiments, the array of light sensing devices comprises one ormore device features selected from the group consisting of: (i) asurface coating to promote adhesion of specific biological, chemical, orphysical entities (e.g., ZrO₂, silane, or thiols); (ii) a surfacecoating to prevent nonspecific binding of specific biological, chemical,or physical entities (e.g., phosphate, phosphonate, PEG-silane, orPEG-thiols); (iii) a differential surface coating to promote binding ofa first type of biological, chemical, or physical entities in somelocations and to prevent non-specific binding in other locations; (iv) asingle-layer surface coating; (v) a multiple-layer surface coating; (vi)a surface coating deposited by atomic layer deposition (ALD), molecularlayer deposition (MLD), chemical layer deposition (CVD), physical layerdeposition (PLD) (e.g., evaporation), spin coating, dipping, or acombination thereof; (vii) a surface coating patterned by lithographyand/or etching processes; (viii) a surface coating with one or moreoptical properties (e.g., bandpass filters, polarization filters,anti-reflection, fluorescent, or reflective coatings); (ix) acompartment of each pixel with nanowell-like structures to preventcross-talk (e.g., opaque walls); (x) a compartment of each pixel withnanowell-like structures to increase fluorescent light collection (e.g.,photo-sensitive walls); and (xi) a combination thereof.

In some embodiments, the array of light sensing devices comprises one ormore flow cells (e.g., fabricated directly on top of the array of lightsensing pixels).

In some embodiments, the array of light sensing devices comprises one ormore instruments selected from the group consisting of: (i) aninstrument configured for detection of an array of immobilizedbiological, chemical, or physical entity without scanning a detector ofthe instrument; (ii) an instrument configured for detection of an arrayof immobilized biological, chemical, or physical entity without any lensof a detector of the instrument; (iii) an instrument configured fordetection of an array of immobilized biological, chemical, or physicalentity without a focusing mechanism of a detector of the instrument;(iv) an instrument configured for parallel excitation of immobilizedfluorescent markers (e.g., configured to use four-beam interference tocreate a two-dimensional sine wave pattern); and (v) a combinationthereof.

In some embodiments, the array of light sensing devices is made ofmaterial compatible with complementary metal-oxide semiconductor (CMOS)processing, and wherein the array of light sensing devices is configuredto be functionalized.

In some embodiments, the array of light sensing devices is fabricatedusing one or more process steps selected from the group consisting of:(i) differential functionalization of an active surface of the array oflight sensing devices; (ii) integration of nanowells to preventcross-talk; (iii) integration of nanowells to increase light collection;(iv) assembly of flow cell directly on array of light sensing devices;and (v) a combination thereof.

In some embodiments, a dimension and/or pitch of individual pixels ofthe array of light sensing devices is matched to a dimension and/orpitch of the array of biological, chemical, or physical entities.

In some embodiments, the array of light sensing devices comprises acoating comprising materials selected from the group consisting of: ametal (e.g., gold); a metal oxide (e.g., ZrO₂); and a metal nitride(e.g., TiN).

In some embodiments, the array of light sensing devices comprises asurface chemistry selected from the group consisting of: silanes (e.g.,APTES); phosphates; phosphonates (e.g., (Aminomethyl)phosphonic acid orfree phosphate); and thiols (e.g., Thiol-PEG-Amine or mPEG-Thiol).

In some embodiments, individual pixels of the array of light sensingdevices are surrounded by a microwell or nanowell or other barrierbetween adjacent pixels to prevent crosstalk between pixels and/or toincrease light collection. In some embodiments, the microwell ornanowell comprises walls that are opaque to light at an emissionwavelength of the array of biological, chemical, or physical entities(e.g., a metal, such as Al or Ti). In some embodiments, the microwell ornanowell comprises walls made of one or more layers of material toconvert photons to electrons (e.g., a silicon p-n junction); and/or oneor more layers of material to collect generated electrons (e.g., ametal, such as Al or Ti).

In another aspect, the present disclosure provides a device for on-chipdetection of an array of biological, chemical, or physical entities,comprising (a) an array of light sensing devices; and (b) an array ofbiological, chemical, or physical entities, wherein the array ofbiological, chemical, or physical entities is immobilized on a substrateof the array of light sensing devices; wherein the array of lightsensing devices is configured to acquire pixel information of the arrayof biological, chemical, or physical entities without scanning the arrayof light sensing devices across the array of biological, chemical, orphysical entities.

In some embodiments, the array of biological, chemical, or physicalentities comprises biological, chemical, or physical entities selectedfrom the group consisting of: (i) a single structured nucleic acidparticle (SNAP); (ii) a single SNAP with at least one fluorescent label;(iii) a DNA origami; (iv) a DNA origami with at least one fluorescentlabel; (v) a single protein (antibody, antigen, peptide, aptamer, orother proteins); (vi) a single protein (antibody, antigen, peptide,aptamer, or other proteins) bound to a single SNAP; (vii) a singleprotein (antibody, antigen, peptide, aptamer, or other proteins) boundto a single DNA origami, one or more fluorescent probes bound to abiological, chemical, or physical entity of (i)-(vii); (ix) one or morenanoparticles (e.g., organic, inorganic, or biological); (x) one or morenanoparticles with optical properties (e.g., quantum dots); (xi) one ormore formulations of dendrimers; and (xii) a combination thereof.

In some embodiments, the array of light sensing devices comprises one ormore device features selected from the group consisting of: (i) asurface coating to promote adhesion of specific biological, chemical, orphysical entities (e.g., ZrO₂, silane, or thiols); (ii) a surfacecoating to prevent nonspecific binding of specific biological, chemical,or physical entities (e.g., phosphate, phosphonate, PEG-silane, orPEG-thiols); (iii) a differential surface coating to promote binding ofa first type of biological, chemical, or physical entities in somelocations and to prevent non-specific binding in other locations; (iv) asingle-layer surface coating; (v) a multiple-layer surface coating; (vi)a surface coating deposited by atomic layer deposition (ALD), molecularlayer deposition (MLD), chemical layer deposition (CVD), physical layerdeposition (PLD) (e.g., evaporation), spin coating, dipping, or acombination thereof; (vii) a surface coating patterned by lithographyand/or etching processes; (viii) a surface coating with one or moreoptical properties (e.g., bandpass filters, polarization filters,anti-reflection, fluorescent, or reflective coatings); (ix) acompartment of each pixel with nanowell-like structures to preventcross-talk (e.g., opaque walls); (x) a compartment of each pixel withnanowell-like structures to increase fluorescent light collection (e.g.,photo-sensitive walls); and (xi) a combination thereof.

In some embodiments, the array of light sensing devices comprises one ormore flow cells (e.g., fabricated directly on top of the array of lightsensing pixels, or assembled/prepared postfabrication).

In some embodiments, the array of light sensing devices comprises one ormore instruments selected from the group consisting of: (i) aninstrument configured for detection of an array of immobilizedbiological, chemical, or physical entity without scanning a detector ofthe instrument; (ii) an instrument configured for detection of an arrayof immobilized biological, chemical, or physical entity without any lensof a detector of the instrument; (iii) an instrument configured fordetection of an array of immobilized biological, chemical, or physicalentity without a focusing mechanism of a detector of the instrument;(iv) an instrument configured for parallel excitation of immobilizedfluorescent markers (e.g., configured to use four-beam interference tocreate a two-dimensional sine wave pattern); and (v) a combinationthereof.

In some embodiments, the array of light sensing devices is made ofmaterial compatible with complementary metal-oxide semiconductor (CMOS)processing, and wherein the array of light sensing devices is configuredto be functionalized.

In some embodiments, the array of light sensing devices is fabricatedusing one or more process steps selected from the group consisting of:(i) differential functionalization of an active surface of the array oflight sensing devices; (ii) integration of nanowells to preventcross-talk; (iii) integration of nanowells to increase light collection;(iv) assembly of flow cell directly on array of light sensing devices;and (v) a combination thereof.

In some embodiments, a dimension and/or pitch of individual pixels ofthe array of light sensing devices is matched to a dimension and/orpitch of the array of biological, chemical, or physical entities.

In some embodiments, the array of light sensing devices comprises acoating comprising materials selected from the group consisting of: ametal (e.g., gold); a metal oxide (e.g., ZrO₂); and a metal nitride(e.g., TiN).

In some embodiments, the array of light sensing devices comprises asurface chemistry selected from the group consisting of: silanes (e.g.,APTES); phosphates; phosphonates (e.g., (Aminomethyl)phosphonic acid orfree phosphate); and thiols (e.g., Thiol-PEG-Amine or mPEG-Thiol).

In some embodiments, individual pixels of the array of light sensingdevices are surrounded by a microwell or nanowell to prevent crosstalkbetween pixels and/or to increase light collection. In some embodiments,the microwell or nanowell comprises walls that are opaque to light at anemission wavelength of the array of biological, chemical, or physicalentities (e.g., a metal, such as Al or Ti). In some embodiments, themicrowell or nanowell comprises walls made of one or more layers ofmaterial to convert photons to electrons (e.g., a silicon p-n junction);and/or one or more layers of material to collect generated electrons(e.g., a metal, such as Al or Ti).

In some embodiments, the present disclosure provides a systemcomprising: (a) a device of the present disclosure; and (b) anon-transitory computer-readable storage medium comprisingmachine-executable code that, upon execution by one or more computerprocessors, implements a method for on-chip detection of an array ofbiological, chemical, or physical entities, the method comprising: (i)using the array of light sensing devices, acquiring pixel information ofthe array of biological, chemical, or physical entities without scanningthe array of light sensing devices across the array of biological,chemical, or physical entities; and (ii) detecting the array ofbiological, chemical, or physical entities based at least in part on theacquired pixel information.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates an example workflow of a method for performingsingle-molecule detection using fabricated integrated on-chip devices,in accordance with disclosed embodiments.

FIG. 2 illustrates a plot for transmission of a fluorescein or Alexa 488emission filter vs. wavelength (nm) in a dry environment, in accordancewith disclosed embodiments.

FIG. 3 illustrates a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

FIG. 4 illustrates a top view of an array of light-sensing devices withfluorescently labeled biomolecules bound to a functionalized area of thechip surface (e.g., immobilized SNAPs) and a probe, in accordance withdisclosed embodiments.

FIG. 5 illustrates a cross-sectional view of one pixel of alight-sensing device with fluorescently labeled biomolecules bound to afunctionalized area of the chip surface (e.g., immobilized SNAPs) and aprobe, in accordance with disclosed embodiments.

FIG. 6 illustrates a cross-sectional view of one pixel of alight-sensing device with fluorescently labeled biomolecules bound to afunctionalized area of the chip surface (e.g., immobilized SNAPs), aprobe, and a differential surface coating, in accordance with disclosedembodiments.

FIG. 7 illustrates a cross-sectional view of one pixel of alight-sensing device with fluorescently labeled biomolecules bound to afunctionalized area of the chip surface (e.g., immobilized SNAPs), aprobe, and a micro-well to prevent cross talk between pixels, inaccordance with disclosed embodiments.

FIG. 8 illustrates a cross-sectional view of one pixel of alight-sensing device with fluorescently labeled biomolecules bound to afunctionalized area of the chip surface (e.g., immobilized SNAPs), aprobe, and a micro-well to increase collection and conversion of emittedlight, in accordance with disclosed embodiments.

FIGS. 9A, 9B, and 9C depict various surface densities of single moleculebiomolecule arrays configured over detection pixels, in accordance withdisclosed embodiments.

FIG. 10 illustrates a cross-sectional view of an array of biomoleculescoupled to a solid support with integrated light-sensing devices, withdetectable affinity reagents coupled to some of the biomolecules, inaccordance with disclosed embodiments.

DETAILED DESCRIPTION OF THE INVENTION

Biological assays may be used for applications such as genome sequencingor protein expression. It may be beneficial to tailor the design ofbiological assays for the fast, high-confidence identification of alarge number of small amounts of different biological materials.However, such requirements may introduce challenges in the form ofcompeting constraints on the design and fabrication of chips, flowcells, and detection systems used for such assays. For example, thelarge number of objects to be detected may impose constraints on theamount of material that can be used for each object and on the densityat which these objects can be loaded on a substrate of reasonable size.These limitations in turn may imply that only limited amount of signalmay be available for detection and that the signal of neighboringobjects becomes difficult to differentiate. Further, the limited signalamount emitted by each object can negatively impact a detection timeneeded to detect the objects.

Thanks to its many advantages (variety of probes, simple bindingmechanism, optical detection), fluorescent labeling became the method ofchoice for many bio-assays. Often, the particles or molecules to bedetected are immobilized on a flat substrate and detection may beperformed with a fluorescent microscope. However, this approach may belimited by the microscope resolution, the intensity of the fluorescentsignal emitted by the label, the cross talk between the signals ofneighboring objects, the noise level and the time needed to scan largearrays of immobilized objects.

The present disclosure provides methods and systems for performingsingle-molecule detection using fabricated integrated on-chip devices.Using disclosed methods and systems, single-molecule detection can beperformed while achieving advantages such as: a reduction in thescanning time required by using a large number of light sensors forparallel imaging without moving parts during imaging, a reduction innoise levels by reducing the number of components in the imaging system,an improved resolution arising from detecting one object on each sensor,decreased crosstalk between neighboring object signals, and improveddetection sensitivity arising from improved light collection enabled bymicroscale or nanoscale features on the imaging sensors.

In assays with fluorescent detection, one or more objects (often verylarge arrays of them) may be immobilized on a surface, and this surfacemay be scanned with a microscope to detect any fluorescent signal fromthe immobilized objects. The microscope itself may comprise a digitalcamera configured to record, store, and analyze the data collectedduring the scan. These cameras may comprise an array of light sensingdevices, such as charge coupled device (CCD) sensors, complementarymetal-oxide-semiconductor (CMOS) sensors, charge injection device (CID)sensors, or JOT image sensors (Quanta).

Using systems and methods of the present disclosure, the objects to beimaged may be immobilized directly on the surface of the array of lightsensing devices. Since such devices may be made out of CMOS-compatiblematerials, their imaging side can be differentially functionalized, andbiological, chemical, or physical entities can then be bound to specificlocations. In some embodiments, one biological, chemical, or physicalentity to be detected is bound on each light sensing device (pixel) ofsuch an array.

Using systems and methods of the present disclosure, the light pathbetween the object to be imaged and the light sensing device can beadvantageously reduced, thereby reducing the noise and distortionscreated along this light path by optical or flow cell components.

Using systems and methods of the present disclosure, each pixel of thelight sensing array can be advantageously used to image one object. Incomparison, for example, due to resolution limits, a camera used in amicroscope may be expected to use at least four pixels per object. Insome embodiments, each pixel may have a size of, for example, about 100nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600nm, about 700 nm, about 800 nm, about 900 nm, about 1 μm, about 1.2 μm,about 1.5 μm, about 1.7 μm, about 2 μm, about 2.5 μm, about 3 μm, about3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 6.5 μm, about 7.4μm, about 8 μm, about 9 μm, about 10 μm, or about 11 μm. The lightsensing array may have a size of about 100 kilopixels, about 200kilopixels, about 300 kilopixels, about 400 kilopixels, about 500kilopixels, about 600 kilopixels, about 700 kilopixels, about 800kilopixels, about 900 kilopixels, about 1 megapixels, about 1.2megapixels, about 1.4 megapixels, about 1.6 megapixels, about 1.8megapixels, about 2 megapixels, about 2.5 megapixels, about 3megapixels, about 3.5 megapixels, about 4 megapixels, about 5megapixels, about 6 megapixels, about 8 megapixels, about 10 megapixels,about 15 megapixels, about 20 megapixels, about 30 megapixels, about 50megapixels, about 100 megapixels, about 200 megapixels, about 500megapixels, about 1 gigapixel, about 2 gigapixels, about 5 gigapixels,or about 10 gigapixels.

Using systems and methods of the present disclosure, the substrate onwhich the biological, chemical, or physical entities are immobilized maynot need to be scanned, thereby saving time, operation costs, and wearon the expensive parts of the instrument.

Referring to FIG. 1 , in an aspect, the present disclosure provides amethod 100 for on-chip detection of an array of biological, chemical, orphysical entities, comprising: providing an array of light sensingdevices (as in step 102); immobilizing the array of biological,chemical, or physical entities on a substrate of the array of lightsensing devices (as in step 104); exposing the array of biological,chemical, or physical entities to electromagnetic radiation sufficientto excite the array of biological, chemical, or physical entities,thereby producing an emission signal of the array of biological,chemical, or physical entities (as in step 106); using the array oflight sensing devices, acquiring pixel information of the emissionsignal of the array of biological, chemical, or physical entitieswithout scanning the array of light sensing devices across the array ofbiological, chemical, or physical entities (as in step 108); and (d)detecting the array of biological, chemical, or physical entities basedat least in part on the acquired pixel information (as in step 110).

Methods and systems of the present disclosure may comprise or beconfigured to allow immobilization of one or more biological, chemical,or physical entities on at least one pixel of a light sensor array. Forexample, biological, chemical, or physical entities may be selectedfrom: (i) a single structured nucleic acid particle (SNAP); (ii) asingle SNAP with at least one fluorescent label; (iii) a DNA origami;(iv) a DNA origami with at least one fluorescent label; (v) a singleprotein (antibody, antigen, peptide, aptamer, or other proteins); (vi) asingle protein (antibody, antigen, peptide, aptamer, or other proteins)bound to a single SNAP; (vii) a single protein (antibody, antigen,peptide, aptamer, or other proteins) bound to a single DNA origami, oneor more fluorescent probes bound to a biological, chemical, or physicalentity of (i)-(vii); (ix) one or more nanoparticles (e.g., organic,inorganic, or biological); (x) one or more nanoparticles with opticalproperties (e.g., quantum dots); (xi) one or more formulations ofdendrimers; and (xii) a combination thereof.

Methods and systems of the present disclosure may comprise one or moredevice features. For example, the one or more device features may beselected from: (i) a surface coating (e.g. ZrO₂, silane, or thiols) topromote adhesion of specific biological, chemical, or physical entities;(ii) a surface coating (e.g. phosphate or phosphonate, PEG-silane, orPEG-thiols) to prevent nonspecific binding of specific biological,chemical, or physical entities; (iii) a differential surface coating topromote binding of a first type of biological, chemical, or physicalentities in some locations and to prevent non-specific binding in otherlocations; (iv) a single-layer surface coating; (v) a multiple-layersurface coating; (vi) a surface coating deposited by atomic layerdeposition (ALD), molecular layer deposition (MLD), chemical layerdeposition (CVD), physical layer deposition (PLD) (e.g., evaporation),spin coating, dipping, or a combination thereof; (vii) a surface coatingpatterned by lithography and/or etching processes; (viii) a surfacecoating with one or more optical properties (e.g., bandpass filters,polarization filters, anti-reflection, fluorescent, reflectivecoatings); (ix) a compartment of each pixel with nanowell-likestructures to prevent cross-talk (nanowells with opaque walls) and/orincrease fluorescent light collection (nanowells with photo-sensitivewalls); and (x) a combination thereof.

Methods and systems of the present disclosure may comprise one or moreflow cells. For example, the one or more flow cells may comprise a flowcell fabricated directly on top of an array of light sensing pixels.

Methods and systems of the present disclosure may comprise one or moreinstruments. For example, the one or more instruments may be selectedfrom: (i) an instrument configured for detection of an array ofimmobilized biological, chemical, or physical entity without scanning adetector of the instrument; (ii) an instrument configured for detectionof an array of immobilized biological, chemical, or physical entitywithout any lens of a detector of the instrument; (iii) an instrumentconfigured for detection of an array of immobilized biological,chemical, or physical entity without a focusing mechanism of a detectorof the instrument; (iv) an instrument configured for parallel excitationof immobilized fluorescent markers (e.g., configured to use four-beaminterference to create a two-dimensional sine wave pattern); and (v) acombination thereof.

As an example, methods and systems of the present disclosure maycomprise immobilization of SNAPs on 300-nm functionalized spots with a1.625-μm pitch. The dimensions of the functionalized spots and/or thepitch may be chosen, for example, to be close to the dimensions ofsuitable image sensing arrays (e.g., commercially available imagesensing arrays). In some embodiments, surfaces of sensing arrays areable to be functionalized because they are made of material compatiblewith complementary metal-oxide semiconductor (CMOS) processing.

Methods and systems of the present disclosure may comprise one or moreprocess steps. For example, the one or more process steps may beselected from: (i) differential functionalization of an active surfaceof the array of light sensing devices; (ii) integration of nanowells toprevent cross-talk; (iii) integration of nanowells to increase lightcollection; (iv) assembly of flow cell directly on array of lightsensing devices; and (v) a combination thereof.

The dimensions of each individual pixel of the light sensing device,which may be a commercially available device, may match the dimensionsof the arrays (e.g., SNAP arrays) on the chips quite well. For example,a typical pixel may have an area of 1.4 μm x 1.4 μm (e.g., 14 megapixelscorresponds to 6.6×4.6 mm²). In comparison, the immobilization spots maybe about 0.3 μm in diameter with a pitch of 1.625 μm. The density of thearrays can be increased, for example, by reducing the pitch to 0.975 μmor even 0.650 μm, but the size of the pixel on commercially availablelight sensing devices may also expect to be reduced in the future. Inprinciple, this design may be extended to much larger sensor arrays,including those with hundreds or even thousands of megapixels (e.g., 100megapixels to 1 gigapixels such as the Canon 120MXS CMOS sensor).

The light sensing devices may acquire image or pixel information at animaging rate of, for example, about 0.1, about 0.5, about 1, about 2,about 3, about 4, about 5, about 10, about 20, about 30, about 40, about50, about 60, 70, 80, 90, about 100, about 200, about 300, about 400,about 500, about 600, about 700, about 800, about 900, about 1000, about1100, about 1200, about 1300, about 1400, about 1500, about 1600, about1700, about 1800, about 1900, about 2000, about 2500, about 5000, about7500, about 10000, or about 20000 frames per second frames per second(fps). The light sensing devices may perform signal amplification, suchas by using one or two amplifiers for each pixel. The signalamplification may be performed by components of the light sensingdevices without using a separate amplification circuit, or by using aseparate amplification circuit, or by a combination thereof. The arrayof light sensing devices may comprise for example, sCMOS sensors havingone or two readout circuits per column of pixels.

A typical coating used on the immobilization spots, or between them, mayinclude one or more dielectrics, one or more plastics, one or more typesof glass, one or more nitrides, one or more metals (e.g., gold), one ormore metal oxidex (e.g. ZrO₂), and/or one or more metal nitrides (TiN)in layer thicknesses varying from a few angstroms to several nanometers.A total number of coating layers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about12, about 14, about 16, about 18, about 20, about 25, about 30, about40, about 50, about 60, about 70, about 80, about 90, or about 100coating layers may be used.

Typical surface chemistries used on the immobilization spots, or betweenthem, may include silanes (e.g., (3-Aminopropyl)triethoxysilane, APTES),phosphates or phosphonates (e.g., (Aminomethyl)phosphonic acid, freephosphate) and thiols (e.g., Thiol-PEG-Amine, mPEG-Thiol), inthicknesses ranging from a few angstroms to a few nanometers. In somecases, a surface may be coated in a metal or metal oxide (e.g., gold,hafnium, aluminum, Al₂O₃, ZrO₂, TiO₂). Surface ligands or functionalgroups may be applied to surfaces as appropriate based upon the surfacematerial (e.g., silanes for silica or glass, phosphates or phosphonatesfor ZrO₂).

Surfaces, including all areas in physical contact with a fluid maycomprise a functionality, mask, adsorbent, texture, microstructure,capture agent, catalyst, deposit, coating, or other surface alteration.The application of a functionality, mask, adsorbent, texture,microstructure, capture agent, catalyst, deposit, coating, or othersurface alteration may include altering hydrophobicity, alteringhydrophilicity, altering amphipathicity, altering surface tension orsurface energy, altering the physical, chemical, electrical, mechanical,or optical characteristics of the fluidic channel, affecting fluid flowor altering fluid properties, increasing or decreasing heat transfer ormass transfer, capturing or adsorbing species from a fluid, preventingadhesion of species from a fluid, performing chemical reactions, andother operations.

In some embodiments, each pixel may be surrounded by a microwell ornanowell to prevent crosstalk between pixels and/or to increase lightcollection. To prevent crosstalk, the wall(s) of these wells maycomprise at least one layer opaque to light (e.g., in a wavelength rangeat which the biological, chemical, or physical entities to be detectedare emitting); an example of such a layer may be a metal (e.g., Al orTi). The layer opaque to light may comprise, for example, a dye. Sincebandpass filter transmission may be a function of angle of incidence, atlarge angles of incidence, the bandpass filter may have low transmissionat the dye's emission wavelengths, thereby reducing crosstalk betweenadjacent pixels. For example, FIG. 2 shows a plot for transmission of afluorescein or Alexa 488 emission filter vs. wavelength (nm) in a dryenvironment, generated using Semrock's “MyLight” software. In water, thedetails may change, but the effect may be similar. The passing band forthe filter may comprise a bandwidth of, for example, about 10 nm, about20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm,about 80 nm, about 90 nm, about 100 nm, about 120 nm, or about 150 nm.In some embodiments, the filters comprise multi-band filters. Thepassing band for the filter may comprise a band center value of, forexample, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about180 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about280 nm, about 300 nm, about 320 nm, about 340 nm, about 360 nm, about380 nm, about 400 nm, about 420 nm, about 440 nm, about 460 nm, about480 nm, about 500 nm, about 520 nm, about 540 nm, about 560 nm, about580 nm, about 600 nm, about 620 nm, about 640 nm, about 660 nm, about680 nm, about 700 nm, about 720 nm, about 740 nm, about 760 nm, about780 nm, about 800 nm, about 820 nm, about 840 nm, about 860 nm, about880 nm, about 900 nm, about 920 nm, about 940 nm, about 960 nm, about980 nm, or about 1,000 nm. The excitation light (e.g., electromagneticradiation sufficient to excite the array of biological, chemical, orphysical entities to produce an emission signal) may have an incidenceangle of about 90 degrees, about 80 degrees, about 70 degrees, about 60degrees, about 50 degrees, about 40 degrees, about 30 degrees, about 20degrees, or about 10 degrees from a surface (e.g., sidewall) of thearray of biological, chemical, or physical entities. To increase lightcollection, these microwell or nanowell walls may contain one or morelayers of material to convert photon to electrons (e.g. a silicon p-njunction) and one or more layers of material to collect the generatedelectrons (e.g., a metal such as Al or Ti).

In some instances, it may be desirable to produce a microarray ornanoarray wherein a plurality of biological, chemical, or physicalentities are spatially distributed over and stably associated with thesurface of a solid support such that each individual biological,chemical, or physical entity may be spatially separated from each otherbiological, chemical, or physical entity.

In some embodiments this disclosure provides methods of producing anarray of spatially separated biological, chemical, or physical entities,a method may comprise: obtaining a solid support with attachment sites,obtaining a sample comprising biological, chemical, or physicalentities, obtaining seeds, each with a functional group, covalentlyattaching each biological, chemical, or physical entity to a single seedvia the functional group, growing each attached seed to a SNAP(Structured Nucleic Acid Particles) of desired size, attaching the SNAPsto the attachment sites of the array, thereby producing a regular arrayof biological, chemical, or physical entities. In some instances, SNAPscan be any type of DNA based nanoparticle, such as rolling circleamplification-based nanoparticles, plasmids, or DNA origaminanoparticles.

For example, methods of producing an array of entities such as proteinsmay begin with the attachment of a protein to an oligonucleotide primervia a linker. The primer can be then annealed to a circular DNAtemplate, and rolling circle amplification can be performed to produce aSNAP (indicated in this example as a DNA cluster). The SNAP can be thendeposited onto a chip. In this example, the negative charge of the DNAbackbone can interact with positively charged features of an array, suchthat the SNAP becomes immobilized on the array.

As another example, methods of producing an array of entities may beginwith a primer having a linker initiating rolling circle amplificationwith a circular DNA template. The resulting SNAP (indicated in thisexample as a DNA cluster) thus comprises a linker, which can then beconjugated to a protein. The SNAP can be then deposited onto a chip. Inthis example, the negative charge of the DNA backbone can interact withpositively charged features of an array, such that the SNAP becomesimmobilized on the array.

As another example, methods of producing an array of entities may beginwith a primer initiating rolling circle amplification with a circularDNA template. The resulting SNAP (indicated in this example as a DNAcluster) can then be joined with a crosslinker, which can then beconjugated with a protein, to result in a SNAP which may be crosslinkedto a protein. The SNAP can be then deposited onto a chip. In thisexample, the negative charge of the DNA backbone can interact withpositively charged features of an array, such that the SNAP becomesimmobilized on the array.

SNAPs may be created, for example by rolling circle amplification orother acceptable method. These SNAPs can be then deposited onto a chip.For example, the negative charge of the DNA backbone can interact withpositively charged features of an array, such that the SNAP becomesimmobilized on the array. Separately, proteins can be modified withchemical handles which can bind a chemical moiety which can be on theSNAPs. The handled proteins can then be applied to the SNAPs, such thatthey covalently attach to the SNAPs.

In some embodiments this disclosure provides arrays of single moleculesand methods and kits for producing arrays of single molecules. In someembodiments this disclosure provides arrays of biological, chemical, orphysical entities and methods and kits for producing arrays ofbiological, chemical, or physical entities. In some examples, an arrayof biological, chemical, or physical entities may comprise an orderedseries of biological, chemical, or physical entities arrayed on a solidsupport. In other examples, an array of biological, chemical, orphysical entities may comprise an irregular array of biological,chemical, or physical entities.

In some examples, biological, chemical, or physical entities on an arraymay be separated by less than 10 nm, about 10 nm, 20 nm, 30 nm, 40 nm,50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1 μm, 1.2μm, 1.4 μm, 1.6 μm, 1.8 μm, 2 μm, 2.5 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm,8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18μm, 19 μm, 20 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 200 μm, 300μm, 400 μm, 500 μm, or more than 500 μm. In some examples, biological,chemical, or physical entities on an array may be separated by at leastabout 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850nm, 900 nm, 950 nm, 1 μm, 1.2 μm, 1.4 μm, 1.6 μm, 1.8 μm, 2 μm, 2.5 μm,3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25 μm, 30 μm, 40 μm, 50μm, 75 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, or more than about500 μm. In some examples, biological, chemical, or physical entities onan array may be separated by no more than 500 μm, 400 μm, 300 μm, 200μm, 100 μm, 75 μm, 50 μm, 40 μm, 30 μm, 25 μm, 20 μm, 19 μm, 18 μm, 17μm, 16 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6μm, 5 μm, 4 μm, 3 μm, 2.5 μm, 2 μm, 1.8 μm, 1.6 μm, 1.4 μm, 1 μm, 950nm, about 900 nm, 850 nm, 800 nm, 750 nm, 700 nm, 650 nm, 600 nm, 550nm, 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 180 nm, 160nm, 140 nm, 120 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30nm, 20 nm, 10 nm, or less than about 10 nm. In some cases, biological,chemical, or physical entities on the array may be separated by betweenabout 50 nm and about 1 μm, about 50 nm and about 500 nm, about 100 nmand about 400 nm, about 200 nm and about 300 nm, about 500 nm and about10 μm, about 50 nm and about 1 μm, or about 300 nm and about 1 μm. Insome cases, the spacing of biological, chemical, or physical entities onthe array may be determined by the presence of attachment sites arrayedon a solid support.

In some embodiments an array may be created on a solid support. Thesolid support may be any solid surface to which molecules can becovalently or non-covalently attached. Non-limiting examples of solidsubstrates include slides, surfaces of elements of devices, surfacecoatings of elements of devices, membranes, flow cells, wells, chambers,and macrofluidic chambers. Solid supports used herein may be flat orcurved, or can have other shapes, and can be smooth or textured. In somecases, solid support surfaces may contain microwells. In some cases,substrate surfaces may contain nanowells. In some cases, solid supportsurfaces may contain one or more microwells in combination with one ormore nanowells. In some embodiments, the solid support can be composedof glass, carbohydrates such as dextrans, plastics such as polystyreneor polypropylene, polyacrylamide, latex, silicon, metals such as gold,chromium, titanium, or tin, titanium oxide, tin oxide, or cellulose. Insome examples, the solid support may be a slide or a flow cell.

A flow cell may be coupled to a solid support. In some embodiments, aflow cell joined to a solid support. In some embodiments, a solidsupport may be incorporated into a flow cell. In some embodiments, asolid support for binding a plurality of attached molecules may bedirectly fabricated on a substrate material (e.g., glass, silica, fusedsilica, quartz). A fabricated substrate may be formed into a flow cellby the enclosure of the solid support area with a cover piece. A flowcell may comprise a fluidic device with one or more ports that permitpassage of fluids into and/or out of the flow cell device. The fluidicdevice may be configured to permit passage of one or more fluids acrossor through the solid support.

Flow cells of the present disclosure may be designed for fluid transferand control at various length scales, including macrofluidic andmicrofluidic length scales. A flow cell may be a particular shapeincluding square, rectangular, oval, or circular. A flow cell may bedesigned to be a fixed or removable piece of a larger fluid transfersystem, with a shape, size, or footprint that may be customized to theneeds of the larger fluid transfer system. A flow cell may have aparticular length, width, or height depending upon the application ofthe fluid. A flow cell may have a length, width, or height of about 1centimeter (cm), 10 cm, 15 cm, 20 cm, 30 cm, 40 cm, 50, or about 100 cm.A fluidic device may have a length, width, or height of at least about 1cm, 10 cm, 15 cm, 20 cm, 30 cm, 40 cm, 50 cm, or about 100 cm or more. Aflow cell may have a length, width, or height of no more than about 100cm, 50 cm, 40 cm, 30 cm, 20 cm, 15 cm, 10 cm, or 1 cm or less.

In some embodiments, a solid support may be characterized by a thicknessor depth. The thickness of a solid support may be uniform or may varyover the body of the solid support. The thickness of the solid supportmay be altered by a fabrication, forming or machining process. In somecases, a solid support may have a thickness of about 1 micrometer (μm),10 μm, 50 μm, 100 μm, 250 μm, 500 μm, 750 μm, 1 millimeter (mm), 5 mm, 1centimeter (cm), 10 cm or more. In some cases, a substrate may have athickness of at least about 1 micrometer (μm), 10 μm, 50 μm, 100 μm, 250μm, 500 μm, 750 μm, 1 millimeter (mm), 5 mm, 1 centimeter (cm), 10 cm ormore. In some cases, a solid support may have a thickness of no morethan about 10 cm, 1 cm, 5 mm, 1 mm, 750 μm, 500 μm, 250 μm, 100 μm, 50μm, 10 μm, 1 μm or less.

In some embodiments, surfaces of the solid support may be modified toallow or enhance covalent or non-covalent attachment of molecules suchas the SNAPs described herein. The solid support and process formolecule attachment are preferably stable for repeated binding, washing,imaging and eluting steps. In some cases, surfaces may be modified tohave a positive or negative charge. In some cases, surfaces may befunctionalized by modification with specific functional groups, such asmaleic or succinic moieties, or derivatized by modification with achemically reactive group, such as amino, thiol, or acrylate groups,such as by silanization. Suitable silane reagents includeaminopropyltrimethoxysilane, aminopropyltriethoxysilane and4-aminobutyltriethoxysilane. The surfaces may be functionalized withN-Hydroxysuccinimide (NHS) functional groups. Glass surfaces can also bederivatized with other reactive groups, such as acrylate or epoxy,using, e.g., epoxysilane, acrylatesilane or acrylamidesilane.

In some embodiments, the solid support may be modified to reducenon-specific attachment of SNAPs to the solid support. In someembodiments, the solid support may be modified to reduce non-specificattachment of biological entities and/or chemical entities to the solidsupport. In some embodiments, the solid support may be passivated. Insome further embodiments, the surface of the solid support may bepassivated. In some embodiments, the passivation layer may includediamond-like carbon, hexa-methyldisilizane, Teflon, fluorocarbon, apolymer such as polyethylene glycol (PEG) and/or Parylene. In someembodiments, a solid support may be passivated by the attachment ofPolyethylene glycol (PEG) molecules across the solid support. In someembodiments, a solid support may be passivated using salmon sperm DNA,glycols, albumin, or a combination of the above. In some embodiments, asolid support may be passivated using one or more components selectedfrom the group consisting of salmon sperm DNA, glycols, and albumin. Insome embodiments, a solid support may be passivated using a blockingreagent such as nitrocellulose or phosphates. In some embodiments,passivation components may be exposed to a surface. In some embodiments,passivation components may not be covalently bound to a surface. In someembodiments, passivation materials may be not covalently bound to thesolid support. In some embodiments, passivating materials may reduce theinstance of non-specific binding of undesired molecules to the solidsupport surface.

Using processes and systems of the present disclosure, surfacefunctionalization and passivation may be performed, which may beadvantageous because functionalizing and passivating molecules can belocalized to specific surface areas based upon a surface material andchemistry. For example, silanated surface functionalizations can bebound to silica surfaces while phosphate passivating groups can be boundto metal oxide surfaces. Surface functionalizations and surfacepassivations may be bound to a surface covalently and may be lesssusceptible to degradation via hydrolysis or other mechanisms. This maylead to more stable and effective coatings in functional and passivatedareas on a surface under a wider variety of conditions and over a longerperiod of time.

Further, the processes provided herein may create coatings ofsubstantially uniform thickness and may be less susceptible topoorly-controlled layer growth. Inorganic, metal, or metal oxidesurfaces may enable precision functional and passivated coatings usingcovalently bound functional groups such as silanes, phosphates, orphosphonates. A number of phosphate or phosphonate-containing molecules(e.g., HPO₃ ²⁻, (aminomethyl)phosphonic acid, PEG-phosphonate) may beeasily deposited from solution or vapor phase on metal or metal oxide(e.g., Au or ZrO₂) surfaces in a self-limited fashion (e.g., formationof self-assembled monolayers, SAMs). Metal or metal oxide/phosphate orphosphonate coatings may be limited to forming SAMs, so processuniformity may be easily controlled. Likewise, silane compounds (e.g.,3-aminopropyl)trimethoxysilane (APTMS), APTES, mercaptosilane) may bedeposited from solution or vapor phase on a substrate such as silica orfused silica in a self-limited fashion.

Certain metals or metal oxides (e.g., Au or ZrO₂) may interact stronglywith phosphates and phosphonates, so processes and systems of thepresent disclosure may also be used to prepare patterned areas ofdirectly-immobilized biomolecules, such as DNA, RNA, phosphopeptides,and phosphoproteins, without the need for additional surfacemodifications after a metal or metal oxide coating may be prepared.Silicon and silica substrates may interact strongly with silanes soprocesses and systems of the present disclosure may also be used toprepare patterned areas of directly-immobilized biomolecules, such asDNA, RNA, phosphopeptides, and phosphoproteins, without the need foradditional surface modifications after the substrate may be prepared.

Through patterning, silane processes (and other material-specificcoating processes) may be compatible with metal or metal oxide-coatedsurfaces. For example, a Si or SiO₂ substrate can be coated with ZrO₂,which can be selectively etched to produce a surface with patternedareas of SiO₂ and ZrO₂. Silane chemistry may be used to selectivelyfunctionalize or passivate the SiO₂ regions, then phosphate orphosphonate chemistry may be used to functionalize or passivate the ZrO₂regions, or vice versa. In some cases, a substrate surface may becompletely functionalized or completely passivated. In other cases,specific areas of a fluidic surface may be functionalized and otherareas may be passivated.

In some embodiments, processes and systems of the present disclosure maycomprise passivation or functionalization for specific target moleculesor particle immobilization. Different passivated or functionalizedregions of metal or metal oxide (e.g., Au or ZrO₂) can be prepared withreagents such as phosphates, phosphonates, and their derivatives. Forexample, passivating or functionalizing using phosphate or phosphonatemay include one or more of: direct immobilization of phosphate- orphosphonate-containing (bio)molecules (e.g., DNA); amine-terminatedphosphates and phosphonates (e.g., (Aminomethyl)phosphonic acid[CAS:1066-51-9]); Aminoalkyl phosphates, phosphonates, or relatedmolecules or compounds with varying alkyl chain length, such as(Aminoethyl)phosphonic acid and (Aminopropyl)phosphonic acid);Carboxy-terminated phosphates and phosphonates, including Carboxyalkylphosphates, phosphonates, or related molecules or compounds, such as(Carboxymethyl)phosphonic acid [CAS:4408-78-0] and relatedcarboxyalkylphosphonates with varying alkyl chain length; Phospholipidsand alkyl-terminated phosphates and phosphonates, such asalkylphosphonic acids, or related molecules or compounds (e.g.,octadecylphosphonic acid (ODPA) [CAS:4724-47-4] and relatedalkylphosphonates with varying alkyl chain length); and thiol-terminatedphosphates and phosphonates, such as Thiophospate [CAS:10489-48-2] orrelated molecules or compounds with varying chain lengths, side groups,and/or compositions.

Different passivated or functionalized regions of silicon, silica, orglass substrates (e.g., fused silica) can be prepared with reagents suchas silanes, organosilanes, and their derivatives. For example,passivating or functionalizing using silanes or organosilanes mayinclude one or more of: amine-terminated silanes (e.g.,(3-aminopropyl)triethoxysilane [CAS:919-30-2];(3-aminopropyl)trimethoxysilane [CAS: 13822-56-5]); amine-terminatedsilanes with secondary amines (e.g., N-(6-aminohexyl)aminomethyltriethoxysilane [15129-36-9]; N-(2-aminoethyl)-3-aminopropyltriethoxysilane [CAS 5089-72-5]; N-(2-aminoethyl)-3-aminopropyltriethoxysilane [CAS 1760-24-3]; halogenated or hydrogenated silanes(e.g., chloro-dimethylsilane [CAS: 1066-35-9]; 4-bromobutyltrimethoxysilane [CAS 226558-82-3]; 7-bromoheptyl trimethoxysilane;5-bromopentyl trimethoxysilane [773893-02-0]; 3-bromopropyltrimethoxysilane [CAS 51826-90-5]; 11-bromoundecyl trimethoxysilane [CAS17947-99-8]; 3-chloroisobutyl trimethoxysilane [17256-27-8];2-(chloromethyl)allyl trimethoxysilane [CAS 39197-94-9] or triethoxysilane [CAS: 2487-90-3]); silanes with alkyl sidechains or varyinglength (e.g., trimethoxypropylsilane [CAS: 1067-25-0]); thiol-terminatedsilanes (e.g., (3-mercaptopropyl)trimethoxysilane [CAS: 4420-74-0]);epoxidated silanes (e.g., 2-(3,4-epoxycyclohexyl)ethyl triethoxysilane[CAS 10217-34-2]; 2-(3,4-epoxycyclohexyl)ethyl trimethoxysilane [CAS3388-04-3]; 5,6-epoxyhexyl triethoxysilane [CAS 86138-01-4];(3-glycidoxypropyl) triethoxysilane [CAS 2602-34-8]; (3-glycidoxypropyl)trimethoxysilane [CAS: 2530-83-8]; 2-(3,4-epoxycyclohexyl)ethylmethyldiethoxysilane [CAS 14857-35-3]; (3-glycidoxypropyl)methyldiethoxysilane [CAS 2897-60-1]; (3-glycidoxypropyl)methyldimethoxysilane [CAS 65799-47-5]; (3-glycidoxypropyl)dimethylethoxysilane [CAS 17963-04-1]; ester-functionalized silanes (e.g.,acetoxymethyl triethoxysilane [CAS 5630-83-1]; acetoxymethyltrimethoxysilane [CAS 65625-39-0]; 2-[(acetoxy(polyethyleneoxy)propyl]triethoxysilane; 3-acetoxypropyl trimethoxysilane [CAS 59004-18-1];benzoyloxypropyl trimethoxysilane [CAS 76241-02-6];10-(carbomethoxy)decyldimethyl methoxysilane [CAS 1211488-83-3];2-(carbomethoxy)ethyl trimethoxysilane;triethoxysilylpropoxy(polyethyleneoxy)dodecanoate [1041420-54-5]; andsilanes with other reactive sidechains (e.g., triethoxy-vinylsilane[CAS: 78-08-0]), or related molecules or compounds with varying chainlengths, side groups, and/or compositions.

A surface functionalization may comprise organic chains containingreactive groups. The reactive groups may be located at a position withina chain, including at a terminal position (e.g., a terminal carboxylicacid group), within a chain (e.g., a secondary amine), or pendant to anatom within a chain (e.g., a non-terminal carboxylic acid group). Insome cases, an organic chain may comprise more than one functional group(e.g., a primary amine and a secondary amine). In some cases, an organicchain may include more than one functional group to create more than onechemical property (e.g., two differing types of reactivity; chemicalreactivity and hydrophobicity). In some cases, a surfacefunctionalization may comprise more than one functional group toincrease the likelihood of a chemical process occurring (e.g., primaryand secondary amines to increase the likelihood of reaction with anamine group).

In some embodiments, processes and systems of the present disclosure maycomprise passivating groups or blocking groups to prevent binding (e.g.,to small molecules, peptides, proteins, nucleic acids, andnanoparticles). Passivating agents may include organic or inorganiccoatings such as metals, metal oxides, and ionic compounds. Surfacepassivating agents may bond with or adsorb to active sites or defects onthe surface of a fabricated substrate, thereby blocking adhesion ofother molecules to active sites or defects. A surface passivating agentmay be applied as a coating, a monolayer or may specifically react atsites that require passivating. A surface passivating agent may beapplied via a liquid or gas phase reaction, or a liquid or gas phasedeposition. A blocking group may include a group that prevents othermolecules from binding to a surface by physically blocking or repellingother molecules from approaching a surface. Blocking molecules mayinclude steric blockers such as branched polymers (e.g., PEG) orlong-chain alkyls. Blocking molecules may include molecules that createrepulsion by electrical or magnetic fields, such as ionic chains orpolymers or magnetic nanoparticles.

Different passivated or blocked regions of a metal or metal oxide (e.g.Au or ZrO₂) can be prepared with reagents such as phosphates,phosphonates, and their derivatives. For example, passivating orblocking using phosphate or phosphonate may include one or more of: freephosphate or hydrogen phosphate, or dihydrogen phosphate;Phosphate-terminated PEG reagents (with various lengths and branching);Bis- or tris-phosphates or phosphonates (di- and tri-phosphonates)(e.g., molecules of varying compositions that have two or more terminalphosphates or phosphonate groups linked by a particular size orcomposition of alkyl, amide, ester, carboxylic acid, alcohol, carbonyl,or other chemical moieties), such as etidronic acid [CAS:25211-86-3] andNitrilotri(methylphosphonic acid) [CAS:6419-19-8]. In some cases, apassivating or blocking group may be deposited in step-wise fashion on ametal or metal oxide surface by first depositing a compound such as aphosphate or phosphonate (e.g., an epoxy-terminated phosphate), thenreacting the epoxy group with a reactive group on a blocking molecule(e.g. an amine-terminated PEG molecule). In other cases, a passivatingagent or blocking group may be deposited in a single step.

Different passivated regions of a silicon, silica, or glass substrates(e.g., fused silica) can be prepared with reagents such as silanes,organosilanes, and their derivatives. For example, passivating usingsilanes or organosilanes may include one or more of: silane (SiH₄) orhalogenated silanes (e.g., SiC₃H); silane-terminated PEG reagents (withvarious lengths and branching); disilanes, trisilanes, or largeroligomerized silanes (e.g., 2 or more bonded silicon atoms withhydrogenated, halogenated, or alkyl side groups); or silanes with alkylside groups (e.g., butylsilane). In some cases, a passivating orblocking group may be deposited in step-wise fashion on an inorganicsubstrate surface such as silicon, silica, or glass substrate, by firstdepositing a compound such as a silane or organosilane (e.g., anepoxy-terminated silane), then reacting an epoxy group with a reactivegroup on a blocking molecule (e.g. an amine-terminated PEG molecule). Inother cases, a passivating agent or blocking group may be deposited in asingle step.

In some embodiments, the solid support may be modified across the entiresurface to which molecules are to be attached. In other embodiments, thesolid support may contain regions which are modified to allow attachmentof molecules and regions which are not modified, or regions which aremodified to decrease attachment of molecules and regions which are notmodified, or regions which are modified to increase attachment ofmolecules and regions which are modified to decrease attachment ofmolecules. In some cases, attachment sites may be created in an array,for example an ordered array.

An ordered array of attachment sites may be created by, for example,photolithography, Dip-Pen nanolithography, nanoimprint lithography,nanosphere lithography, cluster lithography, nanopillar arrays, nanowirelithography, scanning probe lithography, thermochemical lithography,thermal scanning probe lithography, local oxidation nanolithography,molecular self-assembly, stencil lithography, double-beam interferencelithography, or electron-beam lithography. Attachment sites in anordered array may be located such that each attachment site may be lessthan an average of 20 nanometers (nm), or about 20 nm, about 50 nm,about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm,about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm,about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm,about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm,about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm,about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm,about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm,about 950 nm, about 975 nm, about 1000 nm, about 1025 nm, about 1050 nm,about 1075 nm, about 1100 nm, about 1125 nm, about 1150 nm, about 1175nm, about 1200 nm, about 1225 nm, about 1250 nm, about 1275 nm, about1300 nm, about 1325 nm, about 1350 nm, about 1375 nm, about 1400 nm,about 1425 nm, about 1450 nm, about 1475 nm, about 1500 nm, about 1525nm, about 1550 nm, about 1575 nm, about 1600 nm, about 1625 nm, about1650 nm, about 1675 nm, about 1700 nm, about 1725 nm, about 1750 nm,about 1775 nm, about 1800 nm, about 1825 nm, about 1850 nm, about 1875nm, about 1900 nm, about 1925 nm, about 1950 nm, about 1975 nm, about2000 nm, or more than an average of 2000 nm from any other attachmentsite. In some cases, the solid support may comprise a random ornon-ordered array of attachment sites. The spacing of an ordered ornon-ordered array of attachment sites may be calculated as an averagespacing between detected attachment sites as determined by a suitablemethods, such as a method that permits direct detection of attachmentsites (e.g., fluorescent microscopy or surface plasmon resonance) or ananalytical method that permits indirect detection of attachment sites(e.g., spectroscopic quantitation of surface functional groups).

In some cases, the spacing of attachment sites on the solid support maybe selected depending on the size of the SNAPs to be used. For example,the spacing of the attachment sites may be selected such that thedistance between the edges of any two attachment sites may be greaterthan the diameter of the SNAP used.

In some cases, the size of the attachment sites on the solid support maybe selected depending on the size of the SNAPs to be used. For example,the size of the attachment sites may be selected such that the diameterof each attachment sites may be less than the diameter of the SNAP used.

In some cases, the attachment sites may be provided in microwells ornanowells.

In some cases, functional groups may be present in a random spacing andmay be provided at a concentration such that functional groups are onaverage at least about 50 nm, about 100 nm, about 150 nm, about 200 nm,about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm,about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm,about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm,about 1000 nm, or more than 100 nm from any other functional group.

All materials chosen for a solid support may be chosen to exhibitnegligible levels of autofluorescence. Autofluorescence may becharacterized at a particular wavelength or range of wavelengths. Forexample, autofluorescence may be characterized in the ultraviolet,visible, and/or infrared region of the electromagnetic spectrum. Amaterial may be chosen if it has a negligible fluorescence in a rangefrom about 1 nm to 100 nm, 1 nm to 400 nm, 100 nm to 700 nm, 400 nm to700 nm, 400 nm to 1000 nm, 400 nm to 5000 nm, 700 nm to 1000 nm, 700 nmto 5000 nm, or from about 1 nm to about 5000 nm.

In some cases, the solid support may be optically opaque in some cases,the solid support may be optically clear at one or more wavelengths. Insome cases, the solid support may be partially, optically clear, or maybe optically clear in some regions. For example, a solid support may beoptically opaque in regions that are not functionalized, and opticallyclear in regions that are functionalized.

The solid support may be indirectly functionalized. For example, thesolid support may be PEGylated and a functional group may be applied toall or a subset of the PEG molecules.

In some cases, the efficiency of attachment of the SNAPs to the solidsupport may be high. In some cases, the efficiency of attachment of theSNAPs to the solid support may be moderate. In some cases, theefficiency of attachment of the SNAPs to the solid support may be low.The efficiency of the attachment of the SNAPs to the solid support maybe influenced by many factors, including, but not limited to: sequenceof clusters, size of SNAPs relative to size of a corresponding bindingpatch (e.g., large clusters may not bind well to very small patches),the extent to which SNAPs have had their structure modified in such away so as to influence their binding, age of SNAPs, storage conditionsof a buffer or buffers that come into contact with SNAPs, storageconditions of SNAPs, pH or other properties of solvent in which thebinding is hoping to be achieved can massively affect, percentages ofpositive cations, and temperature. In some cases, the reliability ofattachment of the SNAPs to the solid support may be high. In some cases,the reliability of attachment of the SNAPs to the solid support may bemoderate. In some cases, the reliability of attachment of the SNAPs tothe solid support may be low.

In some embodiments, a portion or all of the solid support may beoptically opaque. In some cases, a portion or all of the solid supportmay be optically clear at one or more wavelengths. In some cases, aportion or all of the solid support may be partially optically clear, ormay be optically clear in some regions. For example, an optical coatingon the solid support may be optically opaque in regions that are notfunctionalized, and optically clear in regions that are functionalized.

An example method for producing a solid support and integrated lightsensing devices with attachment sites arrayed at desired intervals maybegin with providing a substrate. In some embodiments, the substrate maybe an array of light sensing devices (e.g., a commercially availablearray of light sensing devices). The substrate may comprise, forexample, a CCD light sensing array, a CMOS devices light sensing array,a light sensing array with a combination of CCD and CMOS devices, acharge injection device (CID) light sensing array, or a JOT imagesensor. In some embodiments, the substrate may be glass. In particular,in some embodiments, the substrate may be amorphous glass, fused silica,or quartz, among other examples. In some embodiments, the substrate maybe silicon. In some embodiments, the thickness of the substrate may beless than 100 microns, 100 microns, 150 microns, 200 microns, 300microns, 400 microns, 500 microns, 600 microns, 700 microns, 800microns, 900 microns, 1 millimeter, 2 millimeters, or more than 2millimeters.

Initially, the substrate may be cleaned, such as with a piranhacleaning. In some embodiments, a substrate may be cleaned using a strongacid so as to clean the substrate without etching the substrate. In someembodiments, the substrate may be cleaned using a detergent.Alternatively, the substrate may be cleaned with solvent, sonication orwith plasma such as O₂ or N₂ plasma, or with a combination thereof.

Once the substrate has been cleaned, a chrome layer may be deposited onthe backside of the substrate. Deposition methods may include, forexample, evaporation or sputtering. In some embodiments, a backsidechrome evaporation may not be applied when a substrate is opaque. Abackside chrome evaporation may have a thickness of one Angstrom, twoAngstroms, 10 Angstroms, 10 nanometers, 20 nanometers, 30 nanometers, 40nanometers, 50 nanometers, 60 nanometers, 70 nanometers, 80 nanometers,90 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250nanometers, 300 nanometers, 400 nanometers, 500 nanometers, or more than500 nanometers. Alternatively, other metals can be used for depositionon the backside of the substrate, such as Aluminum, Tungsten, and/orTitanium, among other examples. Alternatively, dielectric mirrors can beused for deposition on the backside of the substrate.

Further, fiducials may be created on the front side of the substrate.Fiducials may be created by adding at least one layer of material and bypatterning this at least one layer. In some embodiments, such materialcan be chrome, and/or such materials may be other metals like tungstenor gold. Alternatively, dielectric mirrors could be used as a materialfor fiducials. Alternatively, metal oxide could be used for thefiducials as for example ZrO₂. The patterning of such materials can beperformed in a variety of ways. A first way to pattern the fiducialmaterial may be to deposit a blanket layer of the material, then toprotect this material in selected areas and remove the material in theareas where it is not protected. This can for example be achieved bycoating the front side of the substrate with photosensitive material(e.g. photoresist), patterning this photoresist by exposing it to UVlight through a mask and then developing it. The etching of the fiducialmaterial can then be performed by wet etch (for example acid) or dryetch (for example Reactive Ion Etching, RIE). Alternatively, thephotoresist may be deposited and patterned first. In some embodimentswhere the photoresist may be deposited and patterned first, areas aredefined that are free of such photoresist and then the fiducial materialmay be deposited on top of the photoresist. The photoresist may then beremoved (for example, in a solvent bath with sonication) and thefiducial material may be left on the areas that were initially free ofphotoresist (e.g., using a lift-off technique). Alternatively, fiducialsmay be created by removing material from the substrate in selectedareas, for example by patterning a layer of photoresist on the frontside of the substrate and then by dry etching the substrate in the areasthat are not coated with photoresist. In an another alternative,fiducials may be defined by modifying the substrate locally (for exampleby laser melting and/or fractioning). Fiducials may come in a variety ofshapes, lines, and/or orientations. In some embodiments, a pattern offiducials may be applied to the substrate. In yet another embodiment,the shape of fiducials may vary in order to code information about theirlocation on the surface of the substrate.

Once a pattern of fiducials may be created on the front side of thesubstrate, this front side may be differentially coated to definefeatures where the biological objects of interest (for example, nucleicacid clusters covalently attached to a protein) may be immobilized. In afirst embodiment, the surface may be differentially patterned with twosilanes, for example HMDS or a PEG-silane in the field and APTES on theimmobilization spots. This differential patterning may be achieved by,for example, depositing an initial HMDS layer on the surface, followedby a lift-off layer, followed by an optional anti-reflective layer, andfollowed by a photoresist layer. In some embodiments, an anti-reflectivelayer may not be provided when an opaque substrate is being used.

Once the photoresist may be applied, a second lithography step may beprovided. In particular, desired features may be provided. In someembodiments, desired features may have a length of approximately 300 nm.In some embodiments, features may have a length of less than 50 nm, 100nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600nm, 700 nm, or more than 700 nm. In some further embodiments, one ormore layers deposited on the surface to perform this second lithographymay not be etched by the developing step of this second lithography (forexample, the antireflective coating).

In embodiments where a backside coating may be provided, the backsidecoating may be removed, such as through the use of a wet etch or dryetch etc. Further, a directional reactive ion etch (RIE) may be providedso as to remove layers that haven't been removed by the lithography step(for example the antireflective coating).

Once the holes have been provided, cleaning may be performed. As seen inFIG. 2 , an oxygen plasma cleaning and activation step may be provided.Once the chip has been cleaned, an amino-silane deposition may beprovided. Once the amino-silane deposition may be provided, portions ofthe chip manufacture may be lifted-off, such as using hot DMF. Further,a sonication step may be performed. The resulting chip may be used inflow cells for assessments of biological assays.

In an alternative embodiment, the surface may be differentiallypatterned with a silane layer and a metal layer (for example,(3-Aminopropyl)triethoxysilane (APTES) on the immobilization spots andchrome in the field). In another embodiment, the surface may bedifferentially patterned with a silane layer and a metal oxide layer(for example a PEG-silane layer in the field and a ZrO₂ layer on theimmobilization spots). In yet another embodiment, the surface may bedifferentially patterned with a silane layer on the immobilization spots(for example, acyl protein thioesterases (APTS)) and a metal oxide layer(for example a ZrO₂) and a PEG-phosphonic acid layer in the field.

The biological, chemical, or physical entities of this disclosure may beany biological, chemical, or physical entities for which spatialseparation may be desired. In some embodiments, the biological,chemical, or physical entities are proteins. In some cases, the proteinsmay be proteins from a cell or tissue homogenate, from a biologicalfluid, or from an environmental sample. In some cases, the biological,chemical, or physical entities may be antibodies. In some embodimentsthe biological, chemical, or physical entities are nucleic acids. Forexample, the biological, chemical, or physical entities may be DNAs,RNAs, mRNAs, tRNAs, or miRNAs. In some embodiments the biological,chemical, or physical entities are carbohydrates. In some embodiments,the biological, chemical, or physical entities are complex polymers. Insome embodiments the biological, chemical, or physical entities aresmall molecules, for example chemical compounds rather than complexpolymers.

The biological, chemical, or physical entities of this disclosure may beattached to seeds. These seeds are molecules which can be used as astarting ‘seed’ to grow a larger polymeric molecule. The seed may be amonomer capable of being grown into a polymer, or may comprise a monomercapable of being grown into a polymer. Generally, the seeds aremolecules which can be covalently attached to the molecules. The seedsmay have a polarity such that only one functional group of the seed maybe able to bind to a molecule of the molecules to be separated, whileanother one or more functional groups of the seed can form the startingpoint for a polymer.

Examples of monomers which may be present in the seeds include, but arenot limited to, oligonucleotides, carbohydrates, proteins, amyloids,fibrils, and tetratricopeptide repeats. In some cases the seeds aresmall molecules.

The seeds may comprise a monomer and a functional group able to bind toa biological, chemical, or physical entity to be separated. Examples ofsuch functional groups may include, but are not limited to, amines,thiols, carboxylic acids, triple bonds, double bonds, epoxides, alkynes,alkenes, cycloalkynes, azides, cyclo-octynes, cycloalkynes, norbornenes,tetrazines, cycloctanes, epoxides, and hydroxyls. In some cases, theseed may comprise a functional group that is compatible with a clickchemistry. In some cases, the seed may also comprise a linker or spacerbetween the seed and the functional group. In some cases, the linker orspacer may comprise a photo-cleavable bond. In some cases, the seed maycomprise an oligonucleotide conjugated to an amine group on the 5′terminal. In some cases, the seed may comprise an oligonucleotideconjugated to a click chemistry component on the 5′ terminal.

In some cases, bioconjugation may be used to form a covalent bondbetween two molecules, at least one of which may be a biomolecule.Bioconjugation may be formed but not limited to via chemicalconjugation, enzymatic conjugation, photo-conjugation,thermal-conjugation, or a combination thereof. (Spicer, C. D., Pashuck,E. T., & Stevens, M. M., Achieving Controlled Biomolecule-BiomaterialConjugation. Chemical Reviews., 2018, 118, Pgs. 7702-7743, and Greg T.Hermanson, “Bioconjugate Techniques”, Academic Press; 3^(rd) Edition,2013, herein incorporated by reference for this disclosure). In somecases, both the seed and the biological (e.g. SNAP), chemical, orphysical entity may be functionalized. Functionalizing both partners mayimprove the efficiency or speed of a conjugation reaction. For example,a sulfhydryl group (—SH) or amine (—NH₂) of a chemically active site ofa seed, biological, chemical, or physical entity may be functionalizedto allow for greater reactivity or efficiency of a conjugation reaction.Any of a variety of sulfhydryl-reactive (or thiol-reactive) or amineconjugation chemistries may be used to couple chemical moieties tosulfhydryl or amine groups. Examples include, but are not limited to,use of haloacetyls, maleimides, aziridines, acryloyls, arylating agents,vinylsulfones, pyridyl disulfides, TNB-thiols and/or othersulfhydryl-reactive/amine-reactive/thiol-reactive agents. Many of thesegroups conjugate to sulfhydryl groups through either alkylation (e.g.,by formation of a thioether or amine bond) or disulfide exchange (e.g.,by formation of a disulfide bond). More strategies and detail regardingreactions for bioconjugation are described down below and may beextended to other appropriate biomolecules.

Bioconjugation can be accomplished in part by a chemical reaction of achemical moiety or linker molecule with a chemically active site on thebiomolecule. The chemical conjugation may proceed via an amide formationreaction, reductive amination reaction, N-terminal modification, thiolMichael addition reaction, disulfide formation reaction,copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction,strain-promoted alkyne-azide cycloaddition reaction (SPAAC),Strain-promoted alkyne-nitrone cycloaddition (SPANC), inverselectron-demand Diels-Alder (IEDDA) reaction, oxime/hydrazone formationreaction, free-radical polymerization reaction, or a combinationthereof. Enzyme-mediated conjugation may proceed via transglutaminases,peroxidases, sortase, SpyTag-SpyCatcher, or a combination thereof.Photoconjugated and activation may proceed via photoacrylatecross-linking reaction, photo thiol-ene reaction, photo thiol-ynereaction, or a combination thereof. In some cases, conjugation mayproceed via noncovalent interactions, these may be throughself-assembling peptides, binding sequences, host-guest chemistry,nucleic acids, or a combination thereof.

In some cases, site-selectivity methods may be employed to modifyreaction moieties of biomolecules to increase conjugation efficiency,ease of use, reproducibility. Three common strategies are typicallyemployed for site-selective bioconjugation (i) Modification strategiesthat can select a single motif among many, rather than targeting ageneric reactive handle. This may be determined by surrounding asequence, local environment, or subtle differences in reactivity. Theability of enzymes to modify a specific amino acid within a proteinsequence or a glycan at a single position are particularly prominent.Reactions that display exquisite chemo-selectivity also fall within thiscategory, such as those that target the unique reactivity of the proteinN-terminus or the anomeric position of glycans. (ii) The site-specificincorporation of unnatural functionalities, by hijacking nativebiosynthetic pathways may be utilized. (iii) The installation of uniquereactivity via chemical synthesis may be utilized. The complete orpartial synthesis of peptides and oligonucleotides may be widespread,particularly using solid-phase approaches. These techniques allow accessto sequences of up to 100 amino acids or 200 nucleotides, with theability to install a wide variety of functionalized monomers withprecise positional control.

In some cases, chemical conjugation techniques may be applied forcreating biomaterial-biomolecule conjugates. Functional groups used forbioconjugation may be native to the biomolecule or may be incorporatedsynthetically. In the illustrations below, R and R′ may be a biomolecule(for example, but not limited to: SNAP, proteins, nucleic acids,carbohydrates, lipids, metabolites, small molecules, monomers,oligomers, polymers) and/or a solid support.

In some cases, reductive amination may be utilized for bioconjugation.Amines can react reversibly with aldehydes to form a transient iminemoiety, with accompanying elimination of water. This reaction takesplace in rapid equilibrium, with the unconjugated starting materialsbeing strongly favored in aqueous conditions due to the highconcentration of water. However, in a second step the unstable imine canbe irreversibly reduced to the corresponding amine via treatment withsodium cyanoborohydride. This mild reducing reagent enables theselective reduction of imines even in the presence of unreactedaldehydes. As a result, irreversible conjugation of a biomolecule cangradually occur to a biomaterial of interest. In contrast, strongerreducing agents such as sodium borohydride are also able to reducealdehydes. This two-step reductive amination process can also beutilized for the modification of ketones. For example, reductiveamination has therefore been primarily used for the modification ofsodium periodate-treated alginate and chitosan scaffolds. The order ofreactivity may also be reversed for the attachment of reducing sugars,by exploiting the terminal aldehyde/ketone generated in the open-chainform. This strategy, for example, may be exploited to mimic theglucosylation, glycosylation, and/or galactosylation patterns of nativecollagen in ECM, via reductive amination of maltose and lactoserespectively.

In some cases, isothiocyanates of a biomolecule or solid support may beutilized for bioconjugation. For example, isothiocyanate of abiomolecule may react with nucleophiles such as amines, sulfhydryls, thephenolate ion of tyrosine side chains or other biomolecules to form astable bond between two molecules.

In some cases, an isocyanate of a biomolecule or solid support may beutilized for bioconjugation. For example, isocyanates can react withamine-containing molecules to form stable isourea linkages.

In some cases, an acyl azide of a biomolecule or solid support may beutilized for bioconjugation. For example, acyl azide are activatedcarboxylate groups that can react with primary amines to form amidebonds.

In some cases, an amide of a biomolecule or solid support may beutilized for bioconjugation. For example, the use of reactiveN-hydroxysuccinimide (NHS) esters may be particularly widespread. WhileNHS-esters can be preformed, often they are instead generated in situthrough the use of N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide(EDC) coupling chemistry and coupled directly to the species ofinterest. Although formation of the activated NHS-ester may be favoredunder mildly acidic conditions (pH˜5), subsequent amide coupling may beaccelerated at higher pHs at which the amine coupling partner may be notprotonated. One-step modification at an intermediate pH of ˜6.5 may bepossible. Conjugation may be typically undertaken by first forming theactive NETS-ester at pH 5, before raising the pH to ˜8 and adding theamine coupling partner in a two-step procedure. In some cases,water-soluble derivative sulfo-NHS may be utilized as an alternative. Insome cases, NHS esters of a biomolecule can react and couple withtyrosine, serine, and threonine —OH groups as opposed to N-terminalα-amines and lysine side-chain ε-amines.

In some cases, a sulfonyl chloride of a biomolecule or solid support maybe utilized for bioconjugation. For example, reaction of a sulfonylchloride compound with a primary amine-containing molecule proceeds withloss of the chlorine atom and formation of a sulfonamide linkage.

In some cases, a tosylate ester of a biomolecule or solid support may beutilized for bioconjugation. For example, reactive groups comprisingtosylate esters can be formed from the reaction of 4-toluenesulfonylchloride (also called tosyl chloride or TsCl) with a hydroxyl group toyield the sulfonyl ester derivative. The sulfonyl ester may couple withnucleophiles to produce a covalent bond and may result in a secondaryamine linkage with primary amines, a thioether linkage with sulf-hydrylgroups, or an ether bond with hydroxyls.

In some cases, a carbonyl of a biomolecule or solid support may beutilized for bioconjugation. For example, carbonyl groups such asaldehydes, ketones, and glyoxals can react with amines to form Schiffbase intermediates which are in equilibrium with their free forms. Insome cases, the addition of sodium borohydride or sodiumcyanoborohydride to a reaction medium containing an aldehyde compoundand an amine-containing molecule will result in reduction of the Schiffbase intermediate and covalent bond formation, creating a secondaryamine linkage between the two molecules.

In some cases, an epoxide or oxirane of a biomolecule or solid supportmay be utilized for bioconjugation. For example, an epoxide or oxiranegroup of a biomolecule may react with nucleophiles in a ring-openingprocess. The reaction can take place with primary amines, sulfhydryls,or hydroxyl groups to create secondary amine, thioether, or ether bonds,respectively.

In some cases, a carbonate of a biomolecule or solid support may beutilized for bioconjugation. For example, carbonates may react withnucleophiles to form carbamate linkages, disuccinimidyl carbonate, canbe used to activate hydroxyl-containing molecules to form amine-reactivesuccinimidyl carbonate intermediates. In some cases, this carbonateactivation procedure can be used in coupling polyethylene glycol (PEG)to proteins and other amine-containing molecules. In some cases,nucleophiles, such as the primary amino groups of proteins, can reactwith the succinimidyl carbonate functional groups to give stablecarbamate (aliphatic urethane) bonds

In some cases, an aryl halide of a biomolecule or solid support may beutilized for bioconjugation. For example, aryl halide compounds such asfluorobenzene derivatives can be used to form covalent bonds withamine-containing molecules like proteins. Other nucleophiles such asthiol, imidazolyl, and phenolate groups of amino acid side chains canalso react to form stable bonds with a biomolecule or solid support. Insome cases, fluorobenzene-type compounds have been used as functionalgroups in homobifunctional crosslinking agents. For example, theirreaction with amines involves nucleophilic displacement of the fluorineatom with the amine derivative, creating a substituted aryl amine bond.

In some cases, an imidoester of a biomolecule or solid support may beutilized for bioconjugation. For example, the α-amines and ε-amines ofproteins may be targeted and crosslinked by reacting withhomobifunctional imidoesters. In some cases, after conjugating twoproteins with a bifunctional imidoester crosslinker, excess imidoesterfunctional groups may be blocked with ethanolamine.

In some cases, carbodiimides may be utilized for bioconjugation.Generally, carbodiimides are zero-length crosslinking agents that may beused to mediate the formation of an amide or phosphoramidate linkagebetween a carboxylate group and an amine or a phosphate and an amine,respectively. Carbodiimides are zero-length reagents because in formingthese bonds no additional chemical structure may be introduced betweenthe conjugating molecules. In some cases, N-substituted carbodiimidescan react with carboxylic acids to form highly reactive, O-acylisoureaderivatives. This active species may then react with a nucleophile suchas a primary amine to form an amide bond. In some cases, sulfhydrylgroups may attack the active species and form thioester linkages. Insome cases, hydrazide-containing compounds can also be coupled tocarboxylate groups using a carbodiimide-mediated reaction. Usingbifunctional hydrazide reagents, carboxylates may be modified to possessterminal hydra-zide groups able to conjugate with other carbonylcompounds.

In some cases, a biomolecule containing phosphate groups, such as the5′phosphate of oligonucleotides, may also be conjugated toamine-containing molecules by using a carbodiimide-mediated reaction.For example, the carbodiimide of a biomolecule may activate thephosphate to an intermediate phosphate ester similar to its reactionwith carboxylates. In the presence of an amine, the ester reacts to forma stable phosphoramidate bond.

In some cases, an acid anhydride of a biomolecule or solid support maybe utilized for bioconjugation. Anhydrides are highly reactive towardnucleophiles and are able to acylate a number of the importantfunctional groups of proteins and other biomolecules. For example,protein functional groups able to react with anhydrides include but notlimited to the α-amines at the N-terminals, the ε-amine of lysine sidechains, cysteine sulfhydryl groups, the phenolate ion of tyrosineresidues, and the imid-azolyl ring of histidines. In some cases, thesite of reactivity for anhydrides in protein molecules may bemodification of any attached carbohydrate chains. In some cases, inaddition to amino group modification in a polypeptide chain,glycoproteins may be modified at their polysaccharide hydroxyl groups toform esterified derivatives.

In some cases, a fluorophenyl ester of a biomolecule or solid supportmay be utilized for bioconjugation. Flurophenyl esters can be anothertype of carboxylic acid derivative that may react with amines consistsof the ester of a fluorophenol compound, which creates a group capableof forming amide bonds with proteins and other molecules. In some cases,fluorophenyl esters may be: a pentafluorophenyl (PFP) ester, atetrafluorophenyl (TFP) ester, or a sulfo-tetrafluoro-phenyl (STP)ester. In some cases, fluorophenyl esters react with amine-containingmolecules at slightly alkaline pH values to give the same amide bondlinkages as NHS esters.

In some cases, hydroxymethyl phosphine of a biomolecule or solid supportmay be utilized for bioconjugation. Phosphine derivatives withhydroxymethyl group substitutions may act as bioconjugation agents forcoupling or crosslinking purposes. For example, tris(hydroxymethyl)phosphine (THP) and β-[tris(hydroxymethyl)phos-phino] propionic acid(THPP) are small trifunctional compounds that spontaneously react withnucleophiles, such as amines, to form covalent linkages.

In some cases, the thiol reactivity of a biomolecule or solid supportmay be utilized for bioconjugation. For example, the thiol group ofcysteine may be the most nucleophilic functional group found among the20 proteinogenic amino acids. Through careful control of pH, selectivemodification over other nucleophilic residues such as lysine can bereadily achieved. Another example, thiol modification ofoligonucleotides may be used to enable derivatization, though the easewith which alternative reactive handles with enhanced chemicalorthogonality can be installed has limited use forbiomaterial-conjugation. Further, the conjugate addition of thiols toα,β-unsaturated carbonyls, also known as Michael addition, may be usedto form polypeptide conjugates in the fields of tissue engineering,functional materials, and protein modification. In general, reactionrates and conjugation efficiencies are primarily controlled by threefactors and may be modified as needed: (i) the pK_(a) of the thiol; (ii)the electrophilicity of the Michael-acceptor; (iii) the choice ofcatalyst. Regarding (i): the thiolate anion may be the activenucleophile during Michael addition, and the propensity of the thiol toundergo deprotonation may determine thiolate concentration and thusreaction rates. For example, the lower pK_(a) of aromatic thiols, whencompared to their aliphatic counterparts, leads to a higher rate ofreaction rate a weak base may be used to catalyze the. As a result,local structure can significantly alter conjugation efficiency,particularly for polypeptide substrates. The pK_(a) and reactivity ofcysteine containing peptides can be altered significantly throughrational choice of surrounding amino acids, the presence of positivelycharged amino acids, such as lysine and arginine, acts to lower thethiol pK_(a) and thus enhance reactivity. Regarding (ii): theMichael-acceptor becomes more electron deficient it becomes moreactivated toward nucleophilic attack, and thus reaction rates increase.Within the most widely utilized acceptors in the biomaterial field, atrend of reactivity can be generalized as maleimides>vinylsulfones>acrylates>acrylamides>methacrylates. Regarding (iii) Michaeladditions can be accelerated by either basic or nucleophilic catalysis(although both act by increasing the concentration of the activethiolate).

In some cases, the unique nucleophilicity of thiols can be exploited forselective reaction with a number of alternative electrophiles, whichallow efficient and selective biomolecule attachment to be achieved. Forexample, one such group are α-halocarbonyls, with iodoacetamide basedreagents finding particular utility. Higher thiol selectivity may beachieved using less electrophilic bromo and even chloro derivatives,though reactivity may be also drastically reduced. More recently,methylsulfonyl heteroaromatic derivatives have emerged as promisingreagents for thiol-specific conjugation. In other cases, alternativethiol-reactive handles, such as disulfide-bridging pyridazinediones,carbonylacrylic reagents, and cyclopropenyl ketones may be utilized forbioconjugation.

In some cases, sulfhydryl of a biomolecule or solid support may beutilized for bioconjugation. In some cases, three forms of activatedhalogen derivatives can be used to create sulfhydryl-reactive compounds:haloacetyl, benzyl halides, and alkyl halides. In each of thesecompounds, the halogen group may be easily displaced by an attackingnucleophilic substance to form an alkylated derivative with loss of HX(where X is the halogen and the hydrogen comes from the nucleophile).Haloacetyl compounds and benzyl halides typically are iodine or brominederivatives, whereas the halo-mustards mainly employ chlorine andbromine forms. Iodoacetyl groups have also been used successfully tocouple affinity ligands to chromatography supports.

In some cases, a maleimide of a biomolecule or solid support may beutilized for bioconjugation. The double bond of maleimides may undergoan alkylation reaction with sulfhydryl groups to form stable thioetherbonds.

In some cases, an aziridine of a biomolecule or solid support may beutilized for bioconjugation. The highly hindered nature of thisheterocyclic ring gives it strong reactivity toward nucleophiles. Forexample, sulfhydryls will react with aziridine-containing reagents in aring-opening process, forming thioether bonds. The simplest aziridinecompound, ethylenimine, can be used to transform available sulfhydrylgroups into amines. In some cases, substituted aziridines may be used toform homobifunctional and trifunctional crosslinking agents.

In some cases, thiol-maleimide reactions are particularly useful forundertaking conjugation at low concentrations or when requiringextremely high efficiencies due to the value of the biomoleculesubstrate. The use of maleimides in bioconjugation may be furtherenhanced by the ease with which they may be introduced into a wide rangeof scaffold materials, through the modification of amines with thedifunctional reagent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, more commonly referred to by its abbreviationSMCC. For example, this reagent has been widely used to first introducea maleimide reactive handle on a biomaterial of choice and then toenable the attachment of both peptides and growth factors to producebioactive scaffolds.

In some cases, an acryloyl of a biomolecule or solid support may beutilized for bioconjugation. The reactive double bonds are capable ofundergoing additional reactions with sulfhydryl groups. In some cases,the reaction of an acryloyl compound with a sulfhydryl group occurs withthe creation of a stable thioether bond. In some cases, the acryloyl hasfound use in the design of the sulfhydryl-reactive fluorescent probe,6-acryloyl-2-dimethylaminonaphthalene.

In some cases, an aryl group of a biomolecule or solid support may beutilized for bioconjugation with a sulfhydryl group. Although arylhalides are commonly used to modify amine-containing molecules to formaryl amine derivatives, they also may react quite readily withsulfhydryl groups. For example, fluorobenzene-type compounds have beenused as functional groups in homobifunctional crosslinking agents. Theirreaction with nucleophiles involves bimolecular nucleophilicsubstitution, causing the replacement of the fluorine atom with thesulfhydryl derivative and creating a substituted aryl bond. Conjugatesformed with sulfhydryl groups are reversible by cleaving with an excessof thiol (such as DTT).

In some cases, the disulfide group of a biomolecule or solid support maybe utilized for bioconjugation. In some cases, compounds containing adisulfide group are able to participate in disulfide exchange reactionswith another thiol. The disulfide exchange (also called interchange)process involves attack of the thiol at the disulfide, breaking the—S—S— bond, with subsequent formation of a new mixed disulfidecomprising a portion of the original disulfide compound. The reductionof disulfide groups to sulfhydryls in proteins using thiol-containingreductants proceeds through the intermediate formation of a mixeddisulfide. In some cases, crosslinking or modification reactions may usedisulfide exchange processes to form disulfide linkages withsulfhydryl-containing molecules.

In some cases, disulfide bonds may be utilized for bioconjugation. Forexample, the use of disulfide exchange reactions may be favored forintroducing peptides or proteins of interest. The most commonly usedreagents in tissue engineering are based upon reactivepyridylthio-disulfides, which undergo rapid thiol-exchange to releasethe poorly nucleophilic and spectroscopically active 2-mercaptopyridine.Additionally, due to the reversible nature of disulfide bond formation,cleavage can be controlled with temporal precision by the addition ofreducing agents such as dithiothreitol (DTT) or glutathione.

In some cases, a pyridyl dithiol functional group may be used in theconstruction of crosslinkers or modification reagents forbioconjugation. Pyridyl disulfides may be created from available primaryamines on molecules through the reaction of 2-iminothiolane in tandemwith 4,4′-dipyridyl disulfide. For instance, the simultaneous reactionamong a protein or other biomolecule, 2-iminothiolane, and4,4′-dipyri-dyl disulfide yields a modification containing reactivepyridyl disulfide groups in a single step. A pyridyl disulfide willreadily undergo an interchange reaction with a free sulfhydryl to yielda single mixed disulfide product.

In some cases, sulfhydryl groups activated with the leaving group5-thio-2-nitrobenzoic acid can be used to couple free thiols bydisulfide interchange similar to pyridyl disulfides, as describedherein. The disulfide of Ellman's reagent readily undergoes disulfideexchange with a free sulfhydryl to form a mixed disulfide withconcomitant release of one molecule of the chromogenic substance5-sulfido-2-nitroben-zoate, also called 5-thio-2-nitrobenzoic acid(TNB). The TNB-thiol group can again undergo interchange with asulfhydryl-containing target molecule to yield a disulfide crosslink.Upon coupling with a sulfhydryl compound, the TNB group is released.

In some cases, disulfide reduction may be performed usingthiol-containing compounds such as TCEP, DTT, 2-mercaptoethanol, or2-mercaptoethylamine.

In some cases, a vinyl sulfone group of a biomolecule or solid supportmay be utilized for bioconjugation. For example, the Michael addition ofthiols to activated vinyl sulfones to form biomolecule-materialconjugates have been used to demonstrate that cysteine capped peptidescould cross-link vinyl-sulfone functionalized multiarm PEGs to formprotease responsive hydrogels, enabling cell invasion during tissuegrowth. In some cases, in addition to thiols, vinyl sulfone groups canreact with amines and hydroxyls under higher pH conditions. The productof the reaction of a thiol with a vinyl sulfone gives a singlestereoisomer structure. In addition, crosslinkers and modificationreagents containing a vinyl sulfone can be used to activate surfaces ormolecules to contain thiol-reactive groups.

In some cases, thiol-containing biomolecules can interact with metalions and metal surfaces to form dative bonds for bioconjugation. In somecases, oxygen- and nitrogen-containing organic or biomolecules may beused to chelate metal ions, such as in various lanthanide chelates,bifunctional metal chelating compounds, and FeBABE. In addition, aminoacid side chains and prosthetic groups in proteins frequently formbioinorganic motifs by coordinating a metal ion as part of an activecenter.

In some cases, thiol organic compounds may be used routinely to coatmetallic surfaces or particles to form biocompatible layers or createfunctional groups for further conjugation of biomolecules. For instance,thiol-containing aliphatic/PEG linkers have been used to formself-assembled monolayers (SAMs) on planar gold surfaces and particles.

In some cases, a number of alternative coupling systems may be used forbiomolecule functionalization. These include the use of O-nitrophenylesters (which possess reduced stability in aqueous conditions) or1,1′-carbonyldiimidazole (CDI) to form amine-bridging carbamate linkagesrather than amides. Hydrazines can also be used in place of aminesduring EDC/NHS mediated couplings. Hydrazine-functionalized peptides canbe coupled to biomaterials in a single step at pH 5-6. In doing so, adegree of site-selectivity can be achieved over lysine residues present.This approach has been successfully implemented by Madl and co-workersto conjugate reactive groups to alginate hydrogels, enabling indirectfunctionalization with growth factors and adhesion peptides.

In some cases, N-terminal modification of a biomolecule may be utilizedfor bioconjugation. For example, 2-pyridinecarboxaldehyde modifiedacrylamide hydrogels may react specifically with the N-terminus of ECMproteins, forming a cyclic imidazolidinone product with the adjacentamide bond and enabling the orientated display of these keybioinstructive motifs.

In some cases, acrylates, acrylamides, and methacrylates of abiomolecule or solid support may be utilized for bioconjugation. In somecases, thiol-ynes of a biomolecule or solid support may be utilized forbioconjugation.

In some cases, thiol-reactive conjugation such as native chemicalligation (NCL) can be utilized to attach peptides and proteins tobiomaterial scaffolds via peptide bond formation. For example, a peptidehaving a C-terminal thioester reacts with an N-terminal cysteine residuein another peptide to undergo a trans-thioesterification reaction, whichresults in the formation of an intermediate thioester with the cysteinethiol.

In some cases, strong binding of (strept)avidin may be used for thesmall molecule biotin for bioconjugation. In some cases, (strept)avidinand biotin may be attached to a biomolecule or solid support forbioconjugation. In some cases, modification reagents can add afunctional biotin group to proteins, nucleic acids, and otherbiomolecules. In some cases, depending on the functionality present onthe biotinylation compound, specific reactive groups on antibodies orother proteins may be modified to create a (strept)avidin binding site.Amines, carboxylates, sulfhydryls, and carbohydrate groups can bespecifically targeted for biotinylation through the appropriate choiceof biotin derivative. In some cases, photoreactive biotinylationreagents are used to add nonselectively a biotin group to moleculescontaining no convenient functional groups for modification. In somecases, biotin-binding proteins can be immobilized onto surfaces,chromatography supports, microparticles, and nanoparticles for use incoupling biotinylated molecules. In some cases, a series of(strept)avidin-biotin interactions can be built upon each other toutilize the multivalent nature of each tetrameric (strept)avidinmolecule and enhance the detection capability for the target. In somecases, amine-reactive biotinylation reagents that may contain reactivegroups off biotin's valeric acid side chain are able to form covalentbonds with primary amines in proteins and other molecules. In somecases, NHS esters spontaneously react with amines to form amide linkageswhereas carboxylate-containing biotin compounds can be coupled to aminesvia a carbodiimide-mediated reaction using EDC. In some cases,NHS-iminobiotin can be used to label amine-containing molecules with animinobiotin tag, providing reversible binding potential with avidin orstreptavidin. In some cases, Sulfo-NHS-SS-biotin (also known asNHS-SS-biotin) may besulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate, along-chain cleavable biotinylation reagent that can be used to modifyamine-containing proteins and other molecules. In some cases,1-biotinamido-4-[4′-(maleimidomethyl) cyclohexane-carboxamido]butane, abiotinylation reagent containing a maleimide group at the end of anextended spacer arm reacts with sulfhydryl groups in proteins and othermolecules to form stable thioether linkages. In some cases,N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide where thereagent contains a 1,6-diaminohexane spacer group which is attached tobiotin's valeric acid side chain, the terminal amino group of the spacermay be further modified via an amide linkage with the acid precursor ofSPDP to create a terminal, sulfhydryl-reactive group. The pyridyldisulfide end of biotin-HPDP may react with free thiol groups inproteins and other molecules to form a disulfide bond with loss ofpyridine-2-thione.

In some cases, a carboxylate of a biomolecule or solid support may beutilized for bioconjugation. In some cases, diazomethane and otherdiazoalkyl derivatives may be used to label caroxylate groups. In somecases, N,N′-Carbonyl diimidazole (CDI) may be used to react withcarboxylic acids under nonaqueous conditions to form N-acylimidazoles ofhigh reactivity. An active carboxylate can then react with amines toform amide bonds or with hydroxyl groups to form ester linkages. Inaddition, activation of a styrene/4-vinylbenzoic acid copolymer with CDImay be used to immobilize an enzyme lysozyme or other biomoleculethrough its available amino groups to the carboxyl groups on to amatrix.

In some cases, carbodiimides function as zero-length crosslinking agentscapable of activating a carboxylate group for coupling with anamine-containing compound for bioconjugation or a solid support. In somecases, carbodiimides are used to mediate the formation of amide orphosphoramidate linkages between a carboxylate and an amine or aphosphate and an amine.

In some cases, N,N′-disuccinimidyl carbonate or N-hydroxysuccinimidylchloroformate may be utilized in bioconjugation. N,N′-Disuccinimidylcarbonate (DSC) consists of a carbonyl group containing, in essence, twoNHS esters. The compound may be highly reactive toward nucleophiles. Inaqueous solutions, DSC will hydrolyze to form two molecules ofN-hydroxysuccinimide (NHS) with release of one molecule of CO₂. Innonaqueous environments, the reagent can be used to activate a hydroxylgroup to a succinimidyl carbonate derivative. DSC-activated hydroxyliccompounds can be used to conjugate with amine-containing molecules toform stable crosslinked products.

In some cases, sodium periodate can be used to oxidize hydroxyl groupson adjacent carbon atoms, forming reactive aldehyde residues suitablefor coupling with amine- or hydrazide-containing molecules forbioconjugation. For example, these reactions can be used to generatecrosslinking sites in carbohydrates or glyco-proteins for subsequentconjugation of amine-containing molecules by reductive amination.

In some cases, enzymes may be used to oxidize hydroxyl-containingcarbohydrates to create aldehyde groups for bioconjugation. For example,the reaction of galactose oxidase on terminal galactose orN-acetyl-d-galactose residues proceeds to form C-6 aldehyde groups onpolysaccharide chains. These groups can then be used for conjugationreactions with amine- or hydrazide-containing molecules.

In some cases, reactive alkyl halogen compounds can be used tospecifically modify hydroxyl groups in carbohydrates, polymers, andother biomolecules for bioconjugation.

In some cases, an aldehyde or ketone of a biomolecule or solid supportmay be used for bioconjugation. For example, derivatives of hydrazine,especially the hydrazide compounds formed from carboxylate groups, canreact specifically with aldehyde or ketone functional groups in targetbiomolecules. To further stabilize the bond between a hydrazide and analdehyde, the hydrazone may be reacted with sodium cyanoborohydride toreduce the double bond and form a secure covalent linkage.

In some cases, an aminooxy group of a biomolecule or solid support maybe used for bioconjugation. For example, the chemoselective ligationreaction that occurs between an aldehyde group and an aminooxy groupyields an oxime linkage (aldoxime) that has been used in manybioconjugation reactions, as well as in the coupling of ligands toinsoluble supports including surfaces. This reaction may be also quiteefficient with ketones to form an oxime called a ketoxime.

In some cases, cycloaddition reactions may be utilized forbioconjugation. In cycloaddition reactions for bioconjugation, two ormore unsaturated molecules are brought together to form a cyclic productwith a reduction in the degree of unsaturation, these reaction partnersrequired are typically absent from natural systems, and so the use ofcycloadditions for conjugation requires the introduction of unnaturalfunctionality within the biomolecule coupling partner.

In some cases, Copper-Catalyzed Azide-Alkyne Cycloadditions may beutilized for bioconjugation. In some cases, the (3+2) cycloadditionbetween an azide and alkyne proceeds spontaneously at high temperatures(>90° C.), producing a mixture of two triazole isomers. In some cases,this reaction proceeds at room temperature, ambient, oxygenated, and/oraqueous environments. In some cases, for example, the formation ofpeptide-material conjugates by CuAAC, using alkyne-capped peptides toform hydrogels with azide-functionalized PEG. In some cases, CuAAC hasbeen widely used to functionalize scaffolds with alkyne and azidefunctionalized peptides and carbohydrates, in part due to the ease withwhich the amino acids azidolysine and homopropargylglycine can beintroduced by solid-phase peptide synthesis. In some cases, To achievebiomaterial conjugation via CuAAC, the required copper(I) catalyst caneither be added directly, or generated in situ by reduction of aninitial copper(II) complex, most commonly using ascorbic acid. Theaddition of a reducing agent further reduces the sensitivity of theCuAAC ligation to oxygen. Although no additional ligand is necessary fortriazole formation, the addition of tertiary amine based ligands may beused.

In some cases, Strain-Promoted Azide-Alkyne Cycloadditions (SPAAC) maybe utilized for bioconjugation. In some cases, highly strainedcyclooctynes react readily with azides to form triazoles underphysiological conditions, without the need for any added catalyst. Insome cases, in addition to the use of SPAAC for peptide conjugation, anumber of prominent reports have used SPAAC to conjugate proteinsubstrates to cyclooctyne functionalized biomaterials via theintroduction of an unnatural azide motif into the protein couplingpartner. In some cases, for example, this may be achieved by includingmaleimide functionalization of native cysteines present in bonemorphogenetic protein-2 (BMP-2), via enzyme-mediated N-terminalmodification of IFN-γ, or via codon reassignment with the unnaturalamino acid 4-azidophenylalanine in a number of protein substrates. Insome cases, supramolecular host-guest interactions can also be used topromote azide-alkyne cycloaddition. For example, by bringing tworeactive partners into close proximity within the cavity of acucurbit[6]uril host, efficient cycloaddition could be achieved on thesurface of proteins, this strategy may be extended to other appropriatebiomolecules.

In some cases, inverse-electron demand Diels-Alder reactions (IEDDA) maybe utilized for bioconjugation. For example, the inverse-electron demandDiels-Alder (IEDDA) reaction between 1,2,4,5-tetrazines and strainedalkenes or alkynes may be employed. A wide range of suitable derivativesfor undertaking biomolecule conjugation have been reported, for example,a series of increasingly strained (and thus reactive) trans-cyclooctenesmay be utilized. In some cases, functionalized norbornene derivativesmay be utilized for undertaking IEDDA reactions. In some cases,triazines may be utilized. In some cases, spirohexene may be utilized.These strategies may be extended to other appropriate biomolecules. Insome cases, hetero-Diels-Alder cycloaddition of maleimides and furansmay be utilized for bioconjugation. For example, the coupling offuran-functionalized RGDS peptides to maleimide-functionalizedPEG-hydrogels may be utilized, this strategy may be extended to otherappropriate biomolecules. In some cases, furan-functionalized hyaluronicacid hydrogels can be cross-linked with a dimaleimide-functionalizedpeptide via Diels-Alder cycloaddition. MMP-cleavable peptides enable themigration of seeded cancer through the gel.

In some cases, oxime and hydrazone formation may be utilized forbioconjugation. In some cases, the stable attachment of peptides and DNAto biomaterials via hydrazone formation can be achieved via difunctionalcross-linking, this strategy may be extended to other appropriatebiomolecules. In some cases, the attachment of ketone or aldehydemodified green fluorescent protein (GFP) or metallothionein tohydroxylamine-functionalized synthetic polymers may be extended to otherappropriate biomolecules. For example, protein cross-linked hydrogelswere produced through oxime modification at both the protein N- andC-termini.

In some cases, the Diels-Alder reaction consists of the covalentcoupling of a diene with an alkene to form a six-membered ring complexfor bioconjugation.

In some cases, transition metal complexes may be utilized forbioconjugation. The nature of late transition metals may make atransition metal complex well suited to the manipulation of unsaturatedand polarizable functional groups (olefins, alkynes, aryl iodides,arylboronic acids, etc.). For example, Pd(0)-functionalized microspheresmay mediate allyl carbamate deprotections and Suzuki-Miyauracross-coupling in the cytoplasm. In other examples, a ruthenium catalystmay be used to mediate allyl carbamate deprotection of a cagedfluorophore inside living cells. In some cases, applications ofpalladium-based applications in cell culture include copper-freeSonagashira coupling, extracellular Suzuki coupling on the surface of E.coli cells, and conjugation of thiol groups with allyl selenosulfatesalts. In some cases, olefin metathesis may be utilized forbioconjugation. For example, with ruthenium complexes, S-allylcysteinecan be easily introduced into proteins by a variety of methods,including conjugate addition of allyl thiol to dehydroalanine, directallylation of cysteine, desulfurization of allyl disulfide, or metabolicincorporation as a methionine surrogate in methionine auxotrophic E.coli.

In some cases, complex formation with boronic acid derivatives may beused for bioconjugation. For example, boronic acid derivatives are ableto form ring structures with other molecules having neighboringfunctional groups consisting of 1,2- or 1,3-diols, 1,2- or 1,3-hydroxyacids, 1,2- or 1,3-hydroxylamines, 1-2- or 1,3-hydroxyamides, 1,2- or1,3-hydroxyoximes, as well as various sugars or biomolecules containingthese species.

In some cases, enzyme-mediated conjugation may be utilized forbioconjugation. For example, the transglutaminase enzyme familycatalyzes the formation of isopeptide bonds between the primary amine oflysine side chains and the amide bonds of a complementary glutamineresidue, this strategy may be extended to other appropriatebiomolecules. In other cases, peroxidase-mediated conjugation may beutilized for bioconjugation. For example, horse radish peroxidase (HRP)may be utilized to oxidize a wide range of organic substrates such asphenol group of tyrosine to generate a highly reactive radical orquinone intermediate that undergoes spontaneous dimerization, resultingin the formation of an ortho carbon-carbon bond between two tyrosineresidues, this strategy may be extended to other appropriatebiomolecules. In some cases, short peptide tags may be utilized forbioconjugation. These peptide tags may be as short as 5 amino acids longand may be appended to a peptide or protein substrate which allows fortheir subsequent modification.

In some cases, polymerization of low molecular weight monomers may beutilized for bioconjugation. Polymerization may be classified asproceeding via one of two mechanisms, either chain-growth orstep-growth. During chain-growth polymerization, monomers are added atthe “active” end of a growing polymer chain, resulting in the formationof high molecular weight materials even at low conversions. Duringstep-growth polymerizations short oligomer chains couple to formpolymeric species, requiring high conversions in order to reach highmolecular weights. Both techniques can be used to formbiomolecule-polymer conjugates. The polymerization of acrylate andmethacrylate monomers has proven particularly fruitful. For example,acrylate and methacrylate modified peptides and glycans can be readilypolymerized. Similarly, availability of the synthetic oligonucleotidephosphoramidite building block “Acrydite”, free-radical polymerizationremains one of the most common methods through which to form DNA and RNAfunctionalized biomaterials. By undertaking polymerization in thepresence of a comonomer, the density of biomolecule presentation can beeasily tuned, allowing potential difficulties from steric hindrance tobe overcome. Initiation of polymerization can be triggered by a numberof means, including heat, UV and visible light, redox reactions, andelectrochemistry. Acrylate modified proteins can also undergopolymerization to produce functional materials, while retainingbiological activity. In some cases living radical polymerizations (LRPs)may be utilized for bioconjugation. For example, the most commonly usedLRPs for the formation of bioconjugates include atom-transfer radicalpolymerization (ATRP), reversible addition-fragmentation chain transfer(RAFT) polymerization, and nitroxide-mediated polymerization (NMP).

In some cases, photoconjugation may be utilized for bioconjugation. Insome cases, polymerization may be initiated by the production of aradical species, which then propagates through bond formation to createan active polymer chain. The initiation step can be induced via a numberof stimuli, with thermal decomposition, redox activation, andelectrochemical ionization of an initiating species being among the mostcommon. Alternatively, many initiators can be activated vialight-induced photolytic bond breakage (type I) or photoactivatedabstraction of protons from a co-initiator (type II). Photoinitiationoffers the benefits of being applicable across a wide temperature range,using narrow and tunable activation wavelengths dependent on theinitiator used, rapidly generating radicals, and the ability to controlpolymerization by removing the light source. Importantly, the toleranceof polymerizations to oxygen may be greatly enhanced, enablingpolymerization in the presence of cells and tissues. The incorporationof acrylate-functionalized peptides and proteins duringphotopolymerization may be used as a method for producing biomaterialconjugates. Alternatively, the photoinitiated attachment of polypeptidesto pendant vinyl groups on preformed materials has also been widelyreported and more recently used for 3D patterning via two-photonexcitation. A wide range of photoinitiators may be used inphotoconjugation conjugations. For example, but not limited to, Eosin Y,2,2-dimethoxy-2-phenyl-acetophenone, Igracure D2959, lithiumphenyl-2,4,6-trimethylbenzoylphosphinate, and riboflavin may be used asphotoinitiators. Photoinitiators generally absorb light to initiate thephotoreaction processes. In some cases, photoconjugation may utilize aphoto thiol-ene reaction. Thiols can also react with alkenes via afree-radical mechanism. A thiol radical first reacts with an alkene togenerate a carbon-centered radical, which can then abstract a protonfrom another thiol and thus propagate the reaction. Photo thiol-enereactions may be accelerated by electron-rich alkenes, which generateunstable carbon-radical intermediates able to rapidly abstractthiol-hydrogens. Exceptions to this rule are norbornene derivatives, inwhich reactivity may be driven instead by the release of ring strainupon thiol addition. This leads to a general trend in reactivity ofnorbornene>vinyl ether>propenyl>allyl ether>acrylate>maleimide.Norbornenes and allyloxycarbonyls (alloc groups) have been particularlywidely used for peptide/protein-biomaterial functionalization, due tothe almost negligible contribution of chain transfer and their ease ofintroduction during peptide synthesis, respectively. For example, analloc group, typically used as an orthogonal lysine protecting groupduring solid-phase peptide synthesis, may be an efficient photothiol-ene reactive handle. In other examples, norbornene photo thiol-enereactions may be used for the tethering and spatial patterning ofbioactive peptides and growth factor proteins. In addition to the mostcommonly used alloc and norbornene reactive groups, other alkenes havealso been used for biomaterial functionalization. For example, codonreassignment has been used to site-specifically incorporateallyl-cysteine residues into proteins, which can subsequently undergoconjugation through the use of photo thiol-ene reactions. Alternatively,acrylates can undergo mixed-mode photopolymerizations in the presence ofcysteine capped peptides, while allyl disulfide structures have recentlybeen shown to undergo reversible and controlled exchange of conjugatedthiols.

In some cases, aryl azide or halogenate aryl azides of a biomolecule orsolid support may be utilized for bioconjugation.

In some cases, photoreactive group benzophenone may be utilized forbioconjugation.

In some cases, photoreactive group anthraquinone may be utilized forbioconjugation.

In some cases, photo thiol-yne reactions may be utilized forbioconjugation. Most examples of photo thiol-yne reactions haveexploited simple propargyl-ether or -amine reactive handles.

In some cases, photocaging and activation of reactive functionalitiesmay be utilized for bioconjugation. Generally, a transient reactivespecies may be formed whether it be an acrylate or thiol derivedradical. In some cases, photocaging may be used to mask or protect afunctional group until it may be desirable for it to be exposed. In somecases, the most widely utilized cages are based around o-nitrobenzyl andcoumarin chromophores. For example, nitrobenzyl-capped cysteine residuesmay be decaged by irradiation with 325 nm UV light, the released thiolmay then react with maleimide-functionalized peptides via Michaeladdition, to generate a patterned hydrogel able to guide cell migration.In some cases, 6-bromo-hydroxycoumarins may be used for thiol-caging. Insome cases, photoaffinitiy probes may be utilized for bioconjugationwhere a highly reactive intermediate upon irradiation, which then reactsrapidly with the nearest accessible functional group with high spatialprecision. In some cases, the most commonly used are phenylazides,benzophenones, and phenyl-diazirines. In some cases, photocagedcycloadditions may be used. For example, the UV irradiation oftetrazoles has been shown to generate a reactive nitrile-imineintermediate which can undergo rapid cycloaddition withelectron-deficient alkenes such as acrylates or acrylamides. In somecases, the nitrile-imine side-reactivity with thiols may be utilized forsite-specifically conjugate cysteine containing proteins to tetrazolefunctionalized surfaces.

In some cases, noncovalent interactions may be utilized forbioconjugation. In some cases, noncovalent binding plays a vital role incells, controlling biomolecular interfaces and influencingprotein-protein interactions, DNA-DNA complexation, DNA-proteininterfaces, protein localization, and more. In some cases, noncovalentsequences which display a binding affinity for the biomolecule ofinterest, allow for postfabrication modification or for nativebiomolecules to be simply sequestered from the surroundings withinbiological samples. The most commonly used binding sequences are shortpeptides between 7 and 20 amino acids in length, derived from a varietyof sources, including known protein binding domains present in vivo ordetermined through techniques such as phage display. In some cases,short oligonucleotides known as aptamers can also be used to bind avariety of protein substrates, including the cytokines vascularendothelial growth factor (VEGF) and platelet derived growth factor(PDGF), as well as cell surface proteins such as epidermal growth factorreceptor (EGFR). In some cases, binding sequences can also be introducedinto a biomaterial with affinity for native biopolymers, such asheparin. In some cases, by first inducing biopolymer binding, theadsorption of an added or endogenous growth factor or signaling proteinto a biomaterial scaffold can then be controlled. In some cases, bindingaffinity at the amino acid level can also be exploited to enable peptideand protein conjugation to certain biomaterial substrates. For example,the binding of unnatural catechol-based amino acids can be used toinduce binding to metal oxide containing bioglasses and metallicimplants, enabling the bioactivity of these important technologies to beenhanced.

In some cases, self-assembling peptides may be utilized forbioconjugation. For example, native peptides and proteins adopt a seriesof secondary structures, including β-sheets and α-helices, which canboth stabilize individual sequences and control interproteinaggregation. In some cases, self-assembling peptides have been usedextensively to assemble hydrogels and fibrous materials. In many ofthese structures, biological epitopes or functional groups can beappended to some or all of the peptide building blocks during peptidesynthesis, to add the desired bioactivity into the system.Peptide-ligands ranging from simple adhesion motifs, to laminin derivedepitopes, and growth factor mimetics have all been displayed on thesurface of self-assembled fibrils. Alternatively, glycopeptides can beassembled in order to recruit extracellular signaling proteins andgrowth factors, mimic glycosylation patterns within hyaluronic acid, orinvestigate optimal sulfonation ratios in glycosaminoglycan scaffolds.In some cases, self-assembling domains can also be added to full-lengthproteins, leading to the incorporation of pendant functionality duringhydrogel formation. In some cases, the propensity of peptides to formsecondary structures has also been exploited within nonself-assemblingscaffolds. This may be achieved by mixing a self-assembling peptide intoa covalent hydrogel, composed of either a noninteracting polymer such asinterpenetrating networks of PEG or systems where additional chargeinteractions further stabilize the final construct, for example betweenpositively charged peptides and negatively charged alginate gels. As analternative, pendant helical groups can be attached to a covalentmaterial and used to drive the noncovalent attachment of bioactivegroups such as growth factors via self-assembly into coiled-coil triplehelices.

In some cases, host-guest chemistry may be utilized for bioconjugation.For example, the adhesive properties of a β-cyclodextrin modifiedalginate scaffold could be controlled in situ through the addition of aguest naphthyl-functionalized RGDS peptide and by subsequentlyintroducing a non-cell adhesive adamantane-RGES peptide with a higherhost binding constant, dynamic modulation of fibroblast cell attachmentwas enabled. Host-guest interactions between cyclodextrin and naphthyl-or adamantane-functionalized peptides allow alginate functionalization,this may be applied to other appropriate biomolecules.

In some cases, biotin-(strept)avidin may be utilized for bioconjugation.For example, avidin and streptavidin are homotetrameric proteins thatcan simultaneously bind up to four molecules of their small moleculebinding partner biotin. The small size of biotin (with a mass of just244 Da) and the ease with which it can be functionalized via its freecarboxylic acid has led to biotin-(strept)avidin binding findingwidespread use as a means to undertake biomaterial conjugation.Streptavidin-protein fusions can be produced recombinantly and bound tosuitably functionalized surfaces to achieve conjugation. In some cases,biomolecule biotinylation may be undertaken, and this construct may bethen bound to a (strept)avidin functionalized surface. In some cases,this can either be achieved by a direct route, via chemicalpreconjugation of the material with (strept)-avidin, or by exploitingthe tetrameric binding of (strept)avidin to mediate indirectmodification or cross-linking of biotin-functionalized scaffolds.

In some cases, nucleic acids may be utilized for bioconjugation. In somecases, in an analogous fashion to self-assembling peptides, nucleicacids can also form assembled materials themselves, to generate tunableplatforms for the display of biomolecules. In some cases, DNA-taggedpeptides and growth factors can be conjugated to a suitablyfunctionalized biomaterial and used to elicit a desired biologicaleffect on a localized cell population.

Generally, incorporating reactive handles may be utilized forbioconjugation. For example, introducing uniquely reactive motifs intobiomolecule substrates provides a chemical “tag” which allowssingle-site selectivity or specificity to be achieved. In some cases,short peptides and oligonucleotides can typically be produced via solidphase synthesis (SPS). The versatility of organic synthesis allowsdifficulties in reactive handle incorporation to be overcome, with awide range of suitably functionalized amino acids and oligonucleotidesavailable as described herein. In some cases, an alternative approachmay be to introduce unnatural amino acids (UAAs) bearing the desiredreactive handles. This may be achieved via the modification of lysineresidues with amine-reactive derivatives. In some cases, the use ofauxotrophic bacterial strains, which are unable to biosynthesise aparticular amino acid and thus require uptake from the growth media, bystarving the bacteria of the native amino acid and supplementing it witha structurally related unnatural analogue, the bacterial cells can willincorporate the UAA during translation. This technique may be used toinstall azide- and alkyne-based mimics of methionine, leading to theintroduction of reactive handles for undertaking CuAAC and SPAACreactions. Analogous strategies can be used for the incorporation ofunnatural monosaccharides, enabling the remodelling of complex glycans.In some cases, the use of codon reassignment using orthogonal tRNA andtRNA synthetase pairs that selectively recognize and charge an UAAduring translation. In some cases, this may be achieved by reassigningthe amber stop-codon, UAG, by incorporating a tRNA_(CUA)/tRNA synthetasepair from an alternative kingdom into the host cell. This pair may beable to install the desired UAA, while being effectively invisible tothe endogenous cell machinery. As a result, site-directed mutagenesiscan be used to introduce a single TAG codon at the desired position ofthe coding DNA, leading to the singular introduction of the UAA withhigh specificity and selectivity.

In some cases, one or more functional groups may release a reporter whenreacted with another functional group, or with a SNAP or biologicalentity, chemical, or physical entity. Having a reporter released whenthe SNAP and biological, chemical, or physical entity are conjugated mayallow tracking of the reaction. In some cases, it may be possible tomonitor the degree of completion of a SNAP-biological/chemical entityconjugation reaction by monitoring the concentration of free reporter.In some cases, the reporter may fluoresce once released by theconjugation reaction.

In some cases, the biological, chemical, or physical entity may befunctionalized with a linker. In some cases, functionalizing thebiological, chemical, or physical entity with a linker may decreasesteric hindrance. A linker may comprise a rigid or semi-rigid moietywhich can hold the biological, chemical, or physical entity away fromthe SNAP. In some cases, the linker may be a long, moderate or shortlinker. In some cases, the linker may comprise one or more componentselected from PEG, DNA, short carboxyl, carbon chain, peptoid, spacer,and/or glycer, among other examples.

In some cases, the SNAPs, seeds, and/or biological, chemical, orphysical entities may be functionalized using single pot proteomicsmethods. Single pot proteomics methods may result in very highefficiency of functionalization. In some cases, single pot proteomicsmethods may be useful to functionalize biological, chemical, or physicalentities with very low levels of loss of the entities.

In some embodiments, a SNAP may be a polymer which may be grown from theseed. For example if the seed is a DNA oligonucleotide then the SNAP maybe a DNA molecule. In some cases, the SNAP may be a DNA molecule withregions of internal complementarity such that the molecule mayself-hybridize. For example, the SNAP may be a DNA cluster, formed byself hybridization within the molecule. In some cases, the SNAP may beformed from DNA, RNA, L-DNA, L-RNA, LNA, PNA, or a mixture of two ormore different types of nucleic acid. In some cases, the SNAP may have arepeating structure, such as a repeating sequence of nucleotides. Insome cases, the SNAP may be an irregular polymer without a repeatingsequence. For example, the SNAP may comprise a random sequence ofnucleotides.

In some cases, a SNAP may be formed by rolling circle amplification. Aplasmid, or other circular nucleic acid molecule, may be provided as atemplate, together with a primer that binds to the circular nucleic acidmolecule, wherein said primer comprises a functional group on the 5′end. Performing a polymerase chain reaction (PCR) with a sufficientlylong extension step, or merely a polymerase extension reaction, willallow the functionalized primer to bind the circular nucleic acidmolecule and produce a single stranded nucleic acid product. The lengthof the single stranded nucleic acid product may be influenced byaltering the extension time, the polymerase enzyme used, or the reactionconditions. In some cases, the circular nucleic acid template containsregions of internal complementarity, such that the single strandednucleic acid product will contain regions which may self-hybridize. Insome cases, the circular nucleic acid template may be a dsDNA molecule.In some cases, the single stranded nucleic acid product may be an ssDNAmolecule. In some cases, the polymerase used may be a DNA polymerase.

In some cases, a SNAP may be formed by nucleic acid origami, or DNAorigami. DNA origami generally refers to the nanoscale folding of DNA tocreate non-arbitrary two- and three-dimensional shapes at the nanoscale.The specificity of the interactions between complementary base pairs canmake DNA a useful construction material. In some cases, the interactionsbetween different regions may be controlled through design of the basesequences. DNA origami may be used to create scaffolds that hold othermolecules in place or to create structures all on its own.

SNAPs as described herein can include those created via nucleic acidorigami. Commonly, nucleic acid origami can refer to DNA origami, but itcan also refer to RNA origami, origami of a combination of DNA and RNAmolecules, or origami of nucleic acid molecules which can be other thanDNA or RNA, such as a silicon-based nucleic acid, among other examples.Nucleic acid origami can result in a nucleic acid molecule which has anengineered shape. The engineered shape can be a shape which has beenpartially or fully planned. The planning of the shape can compriseplanning or engineering what sections of nucleic acid bind, where asegment of nucleic acid can fold, where a segment of nucleic acid can besingle stranded, where a segment of nucleic acid can be double stranded,where a segment of nucleic acid can be bound to a segment of nucleicacid of the same strand, or where a segment of nucleic acid can be boundto a segment of nucleic acid on another strand. In some cases,non-nucleic acid molecules, such as protein, can be used to encouragenucleic acid into the engineered shape.

Generally, nucleic acid origami can comprise at least one or more longnucleic acid strand and one or more short nucleic acid strands.Commonly, these nucleic acid strands are single stranded, although theycan have segments which can be double stranded. One of the short strandscan comprise at least a first segment which can be complementary to afirst segment of the long strand, as well as a second segment which canbe complementary to a second segment of the long strand. When the shortand long strands are incubated under conditions that can allowhybridization of nucleotides, the shorter oligonucleotide can hybridizewith the longer oligonucleotide. This hybridization can give shape tothe nucleic acid molecule. For example, if the two segments on the firststrand are separated, then these two segments can be brought togetherduring hybridization to create a shape. In some cases, a short strandcan bind to at least 2, 3, 4, 5, or 6 segments which can bind to atleast 2, 3, 4, 5, or 6 complementary segments of the long nucleic acidstrand.

In some cases, a short strand can have one or more segments which can benot complementary to the long strand. In such a case, the segment whichmay be not complementary to the long strand can be at least about 1, 2,3, 4, 5, 10, 15, or 20 nucleotides long.

This process can be performed with at least about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300,or more short nucleic acid strands. These short nucleic acid strands caneach bind to one or more different segments of the long nucleic acidstrand. Each short nucleic acid strand which hybridizes to the longnucleic acid strand can lead to a fold in the long nucleic acid strand.In some cases, the number of short strands can be correlated with thecomplexity of the engineered shape. For example, an engineered shapewith many folds can utilize more short nucleic acid strands than anengineered shape with few folds. An engineered shape can have at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, 250, 300, or more folds.

In some cases, more than one long strand can be incorporated into thenucleic acid origami structure. This can be done for example to increasethe complexity of the engineered shape, to ease the designing orplanning of the engineered shape, to avoid the creating of a shape whichmay be more thermodynamically stable than the desired engineered shape,to make the creation of the engineered shape easier, or to manage costsof creating the engineered shape.

Incorporation of more than one long strand can be accomplished bydesigning the 2 or more long strands such that each strand has at leastone segment that can be complimentary to a segment of the other strand,or by designing the 2 or more long strands such that each has at leastone segment which can be complementary to a region of a short nucleicacid strand, such that both long strands have segments complementary tothe short nucleic acid strand.

Short nucleic acid strands can have complementarity to one long nucleicacid strand or more than one long nucleic acid strand. In some cases, ashort nucleic acid strand can also have complementarity to one or moreshort nucleic acid strands.

The terms “long” and “short” herein are meant to be general terms. Along strand can be longer than a short strand, although in someinstances a long strand can be the same size as a short strand. In somecases, a long strand can be at least about 30, 40, 50, 100, 150, 200,250, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides long.In some cases, a short strand can be at least about 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more nucleotideslong.

An engineered shape can be designed for a specific purpose. For example,an engineered shape can be designed to support a load, encapsulate amolecule, bind a molecule, connect two or more molecules, fit into acavity, bind a protuberance, or other purpose. An engineered shape canany shape, such as oblong, rectangular, round, circular, spherical,flat, textured, smooth, symmetrical, asymmetrical, conical, orirregular. An engineered shape can be a cube, pyramid, box, cage,ladder, or tree.

An engineered shape or SNAP formed via nucleic acid origami as describedabove can be assembled. Assembly can refer to the process by which thenucleic acid strands hybridize to each other to create the engineeredshape.

An engineered shape or SNAP can be spontaneously self-assembling.Self-assembly can occur when long and short oligonucleotides havingregions which can be complimentary are incubated together. Duringspontaneous self-assembly, the nucleotides can hybridize and theengineered shape can be created during incubation without the help of ahelper molecule or catalyst. Such self-assembling can occur underspecific conditions or a range of specific conditions. Conditions whichcan be considered when incubating DNA strands for self-assembly can besalt concentration, temperature, and time.

Sometimes, assembly can utilize or require a catalyst. In such cases,the catalyst can speed up assembly or ensure the assembly results in aparticular desired engineered shape. A catalyst can comprise RNA, DNA,or protein components.

The salt concentration during assembly can be less than 1 M, less than0.5M, less than 0.25 M, less than 0.1M, less than 0.05 M, less than 0.01M, less than 0.005 M, or less than 0.001 M.

The temperature during assembly can be at least room temperature. Insome cases, the temperature during assembly can be at least about 50,60, 70, 80, 85, 90, or 95° C. In some cases, the temperature duringassembly can vary. For instance, the temperature can be increased to atleast about 20, 30, 40, 50, 60, 70, 80, 85, 90, or 95° C. This increasecan ensure the nucleic acid strands do not comprise a secondarystructure prior to assembly. Once the temperature is increased asdescribed, it can be decreased, for example to about 20, 30, 40, 50, 60,70, or 80° C. This decrease in temperature can allow the nucleic acidsto hybridize. In some cases, the decrease in temperature can occur overabout 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 45, or 60 minutes.

Assembly can be performed stepwise. In such cases, a subset of thenucleic acid molecules can be incubated together first. After thesemolecules are allowed to hybridize, one or more additional nucleic acidmolecules can be added and allowed to hybridize. In some cases, two ormore engineered shapes which have been assembled can be incubatedtogether for assembly into a larger engineered shape.

In some cases, assembly can comprise fractal assembly. Fractal assemblycan create a SNAP which can be an array of engineered shapes. Assemblycan occur in stages, which can simplify the design process or ensurecorrect assembly. Such an array can be assembled in at least 1, 2, 3, 4,5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,1000, or more stages. In some cases, the number of stages used cancorrelate with a reduction of spurious interactions. This can be due toa reduction in the total number of possible reactions at any given time.

SNAPs can be assembled into an array which can be at least 3×3, at least5×5, at least 10×10, at least 50×50, at least 100×100, or at least1000×1000 (engineered shapes x engineered shapes).

Each hybridization reaction can take about 10, 20, 30, 40, 50, or 60seconds. In some cases, each hybridization reaction can take about 1, 2,3, 4, 5, 10, 15, 20, 30, 40, 50, or 60 minutes. In some cases, ahybridization reaction can take more than 1 hour.

Nucleic acid origami may be used to preferentially choose how the SNAPwill “land” on the solid support. For example, nucleic acid origami maybe used to construct a SNAP with a landing surface that canpreferentially contact the solid support, A SNAP such as one made vianucleic acid origami can be designed to comprise a region that cancreate steric or electrostatic interactions with the support that caninfluence the orientation of the SNAP on the support. For example, theregion can comprise nucleotides having modifications e.g. to thebackbone of the nucleic acid which can promote interaction between theSNAP and the solid support. In further examples, the region can compriseprotuberances or cavities which can “fit” to cavities or protuberanceson the solid support. In some cases, the support surface can comprisechemical structuring (e.g. nanoparticles or oligonucleotides), clickreagents, or other rationally designed materials that can influence theposition and orientation of SNAP structures, including SNAPs synthesizedvia nucleic acid origami.

Nucleic acid origami can be used to construct a SNAP with a linker whichcan attach a biological, chemical, or physical entity, wherein saidlinker may be positioned relative to the landing surface such that thebiological, chemical, or physical entity can be distal or approximatelydistal to the solid support. The linker may also comprise a region ofdsDNA to force a rigid outpost from the SNAP. In some cases, proteinorigami may also be used.

A surface can have properties such that a SNAP can bind to the surfacein such a way that it can flop or lean. The SNAP can flop or lean to theleft, to the right, to the front, to the back, or to any combination ofsides thereof. The SNAP can flop or lean once and remain in place, or itcan flop freely between sides over time. In some cases, the SNAP canpreferentially flop in one direction over one or more other directions.In some cases, the SNAP can preferentially avoid flopping in aparticular direction.

In some cases, for example, filamentous or stranded molecules, such asnanoparticles or oligonucleotide strands, can be attached to a surface.A SNAP, which can comprise an engineered shape, can comprise one or moremoieties which can bind to a filamentous or stranded molecule, such as adangling single stranded oligonucleotide or nanoparticle. Uponcontacting the surface with such SNAPs, the one or more moieties caninteract with one or more of the filamentous or stranded molecules. Insome cases, the moieties can bind tightly to the filamentous or strandedmolecules. The SNAPs can be removable or non-removable in such cases.

Computational modeling or simulation tools may be employed to design andoptimize oligonucleotide or protein sequences to create particular SNAPstructures.

In some cases, a SNAP may be a nucleic acid plasmid, such as a DNAplasmid. Plasmids may exist in a compact form known as supercoiled DNA.The radii of a supercoiled plasmid may be determined by the plasmidsize—i.e. a plasmid with a longer backbone will form a largersupercoiled entity. In some cases, a SNAP may comprise a plasmid with abackbone of between 5 kb and 150 kb. In some cases, a SNAP may comprisea plasmid with a backbone of between 5 kb and 100 kb. In some cases, aSNAP may comprise a plasmid with a backbone of between 5 kb and 90 kb.In some cases, a SNAP may comprise a plasmid with a backbone of between25 kb and 50 kb. In some cases, a SNAP may comprise a plasmid with abackbone of at least about 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 kb, 85kb, 90 kb, 95 kb, 100 kb, 105 kb, 110 kb, 115 kb, 120 kb, 125 kb, 130kb, 135 kb, 140 kb, 145 kb, or 150 kb. In some embodiments, SNAPs may beimaged using an imaging platform, such as Nanocyte or Leica

In some cases, a SNAP may have a branched structure. For example theSNAP may be a dendrimer. Some examples of dendrimers may be found inNewkome, George R., and Carol D. Shreiner. “Poly (amidoamine),polypropylenimine, and related dendrimers and dendrons possessingdifferent 1→2 branching motifs: an overview of the divergentprocedures.” Polymer 49.1 (2008): 1-173. A dendrimer used with themethods of this disclosure may be a G1, G2, G3, G4, G5, G6, G7, G8, G9,G10, G11, G12, G13, G14, or G15 dendrimer. In some cases, the dendrimermay be higher than a G15 dendrimer, for example dendrimer between G15and G30.

In some embodiments, the SNAP may be a protein, or comprised ofproteins. For example the SNAP may be a protein fibril. The SNAP may becomprised of proteins known to form into fibrils, such as, for example,the tau protein, or portions of the tau protein. A 31 residue portion oftau which assembles into fibrils is described in Stöhr, Jan. et al. “A31-residue peptide induces aggregation of tau's microtubule-bindingregion in cells.” Nature chemistry 9.9 (2017): 874. In some cases, theSNAP may comprise tetratricopeptide repeats. Examples oftetratricopeptide repeats may be found in Blatch, Gregory L., andMichael Lässie. “The tetratricopeptide repeat: a structural motifmediating protein-protein interactions.” Bioessays 21.11 (1999):932-939. Other examples of proteins which may assemble may be found inSpeltz, Elizabeth B., Aparna Nathan, and Lynne Regan. “Design ofprotein-peptide interaction modules for assembling supramolecularstructures in vivo and in vitro.” ACS chemical biology 10.9 (2015):2108-2115.

In some embodiments, the SNAP may be a single molecule. In someembodiments the SNAP may not be a single molecule. In some cases, theSNAP may be assembled from several molecules which bind non-covalently.For example, the SNAP may be formed from two or more nucleic acidmolecules which hybridize together. In another example the SNAP may beformed from two or more protein molecules which assemble together vianon-covalent bonds.

In some embodiments, the SNAPs are between about 50 nm and about 100 umin diameter.

The SNAPs are generally polymeric molecules. These may be grown througha controlled polymerization reaction, a stepwise polymerizationreaction, or a step by step synthesis method. The growth of the SNAPsmay be controlled by the amount of monomers available, the length oftime the reaction may be allowed to proceed, or the number of synthesissteps performed.

Each SNAP may have a diameter of at least about 10 nanometers (nm), orabout 10 nm, about 50 nm, about 75 nm, about 100 nm, about 125 nm, about150 nm, about 175 nm, about 200 nm, about 225 nm, about 250 nm, about275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about900 nm, about 925 nm, about 950 nm, about 975 nm, about 1000 nm, about1025 nm, about 1050 nm, about 1075 nm, about 1100 nm, about 1125 nm,about 1150 nm, about 1175 nm, about 1200 nm, about 1225 nm, about 1250nm, about 1275 nm, about 1300 nm, about 1325 nm, about 1350 nm, about1375 nm, about 1400 nm, about 1425 nm, about 1450 nm, about 1475 nm,about 1500 nm, about 1525 nm, about 1550 nm, about 1575 nm, about 1600nm, about 1625 nm, about 1650 nm, about 1675 nm, about 1700 nm, about1725 nm, about 1750 nm, about 1775 nm, about 1800 nm, about 1825 nm,about 1850 nm, about 1875 nm, about 1900 nm, about 1925 nm, about 1950nm, about 1975 nm, about 2000 nm, about 3000 nm, about 4000 nm, about5000 nm, about 6000 nm, about 7000 nm, about 8000 nm, about 9000 nm,about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about40 μm, about 50 μm, about 75 μm, about 100 μm, about 200 μm, about 300μm, about 400 μm, about 500 μm, or more than about 500 μm. In somecases, the SNAP may have a diameter between about 100 nm and 500 nm,between about 200 nm and about 400 nm, between about 500 nm and about 10μm, or between about 1000 nm and about 10 μm.

In some cases the SNAPs may be covalently attached to the solid supportusing a click chemistry. Generally, the term “click chemistry” is usedto describe reactions that are high yielding, wide in scope, create onlybyproducts that can be removed without chromatography, arestereospecific, simple to perform, and can be conducted in easilyremovable or benign solvents (McKay, C., & Finn M. G. (2014) ClickChemistry in Complex Mixtures Bioorthogonal Bioconjugation vol 21, Issue9, pp 1075-1101; M. G. Meldal, M., & Tornøe, C. W. (2008). Cu-CatalyzedAzide-Alkyne Cycloaddition. Chemical Reviews, 108(8), 2952-3015; Lutz,J., & Zarafshani, Z. (2008). Efficient construction of therapeutics,bioconjugates, biomaterials and bioactive surfaces using azide-alkyne“click” chemistry. Advanced Drug Delivery Reviews, 60(9), 958-970,herein incorporated by reference).

In some cases, the click chemistry reaction may be a CuAAC, SPAAC,SPANC, or as described elsewhere herein. In some cases, the clickchemistry reaction may need a copper source such as, for example, CuSO₄,Cu(O), CuBr(Ph₃P)₃, CuBr, CuBr/Cu(OAc)₂, CuBr₂, [Cu(CH3CN)4]PF6,PS-NMe2:CuI, silica:CuI, (EtO)3P:CuI, CuCl/Pd2(dba)3, CuBF4, CuCl,CuCl2, Cu(AcO)2, Cu(2), TTA:CuSO4, Cu(1) zeolite (USY), Cu(CH3CN)4OTf,CuOTf, Cu(2):bis-batho, or a combination thereof. In some cases a coppersource is not needed for the click chemistry reaction to proceed. Insome cases, the reducing agent of the click chemistry reaction may be,for example, NaAsc, air, ICl, oxygen, N₂, HAsc, TCEP, dithithreitol(DTT), PPh₃, mercaptoethanol, tris(2-carboxyethyl)phosphine (TCEP),TCEPT-hydrochloric acid a combination thereof, or no reducing agent. Insome cases, the solvent of the click chemistry reaction may be, forexample, THF, pyridine, DMSO, DMF, toluene, NMP, acetonitrile, water,tBuOH, iBuOH, EtOH, MeOH, dioxane, dichloromethane, HEPES, NaCl buffer,acetone, PBS, SFM, Tris buffer, borate buffer, PB, TFH, AcOEt, PIPES,urea, acetone, Tris, saline, AllOCO₂Me, TMS-N₃, urea solution,bicarbonate buffer, a combination thereof, or no solution. In somecases, the base of the click chemistry reaction may be, for example,DIPEA, Lut Na2CO3, iPr₂NH, DBU, Et₃N, Et₃N HCl, Et₃NH+-OAc, K₂CO₃, TBAF,CuSO₄, PS-NMe₂, piperidine, a desired pH, or a combination thereof. Insome cases, the ligand of the click chemistry reaction may be, forexample, TBTA, proline, BMAH, Lut, chiral Lig's, pyridine, His, Batho,TTA, Bim, Phen, Bipy, PMDETA, dNbipy, TRMEDA, or a combination thereof.In some cases, the temperature of the click chemistry reaction may be,for example, 0-5° C., 5-15° C., 15-25° C., 20-25° C., 25-35° C., 35-45°C., 45-55° C., 55-65° C., 65-75° C., 75-85° C., 85-95° C., or greater.In some cases, the temperature of the click chemistry reaction may beless than 0° C. In some reactions, the click chemistry reaction may becovered by aluminum foil. In some cases, the click chemistry reactionmay include an acid, for example, trifluoroacetic acid, trichloroaceticacid, or tribromoacetic acid.

In some cases, a crosslinker may be used for conjugation. In some cases,the crosslinker may be a zero-length crosslinker, homobifunctionalcrosslinker, heterobifunctional crosslinker, or a trifunctional crosslinker. Crosslinkers may be incorporated into a biomolecule preformed orin-situ.

In some cases, zero-length crosslinkers mediate the conjugation forbioconjugation by forming a bond containing no additional atoms. Thus,one atom of a molecule may be covalently attached to an atom of a secondmolecule with no intervening linker or spacer. In so conjugationschemes, the final complex may be bound together by virtue of chemicalcomponents that add foreign structures to the substances beingcrosslinked. Carbodiimides may be used to mediate the formation of amidelinkages between carboxylates and amines or phosphoramidate linkagesbetween phosphates and amines and are popular type of zero-lengthcrosslinker that may be used, being efficient in forming conjugatesbetween two protein molecules, between a peptide and a protein, betweenan oligonucleotide and a protein, between a biomolecule and a surface orparticle, or any combination of these with small molecules. In somecases, EDC (or EDAC; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride) may be used for conjugating biomolecules containingcarboxylates and amines. In some cases, CMC, or1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (usually synthesized asthe metho p-toluene sulfonate salt), may be a water-soluble reagent usedto form amide bonds between one molecule containing a carboxylate and asecond molecule containing an amine that may be used as a crosslinkerfor bioconjugation. In some cases, DIC, or diisopropyl carbodiimide maybe used for bioconjugation as a zero-length crosslinker. In some cases,DCC (dicyclohexyl carbodiimide) may be used for bioconjugation as azero-length crosslinker. In some cases, Woodward's reagent K isN-ethyl-3-phenylisoxazolium-3′-sulfonate, a zero-length crosslinkingagent able to cause the condensation of carboxylates and amines to formamide bonds. In some cases, CDI, or N,N′-carbonyl diimidazole may beused for bioconjugation as a zero-length crosslinker. In some cases,Schiff base formation and reductive amination may be used forbioconjugation as a zero-length cross linker.

In some cases, homobifunctional crosslinkers mediate the conjugation forbioconjugation. In some cases, homofictuional NHS esters may be used forbioconjugation. For example, Lomant's reagent[(dithiobis(succinimidylpropionate), or DSP]) is a homobifunctional NHSester crosslinking agent containing an eight-atom spacer 12 Å in length.The sulfo-NHS version of DSP, dithiobis(sulfosuccin-imidylpropionate) orDTSSP, is a water-soluble analog of Lomant's reagent that can be addeddirectly to aqueous reactions without prior organic solvent dissolution.In some cases, disuccinimidyl suberate (DSS), an amine-reactive,homobifunctional, NHS ester, crosslinking reagent produces an eight-atombridge (11.4 Å) between conjugated biomolecules. In some cases,disuccinimidyl tartarate (DST), a homobifunctional NHS estercrosslinking reagent that contains a central diol that is susceptible tocleavage with sodium periodate may be used forms amide linkages withα-amines and ε-amines of proteins or other amine-containing molecules.In some cases, BSOCOES [bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone], a water-insoluble, homobifunctional NHS ester crosslinkingreagent that contains a central sulfone group, where the two NHS esterends are reactive with amine groups in proteins and other molecules toform stable amide linkages. In some cases, ethyleneglycolbis(succinimidylsuccinate) (EGS), a homobifunctional crosslinkingagent that contains NHS ester groups on both ends. The two NHS estersare amine reactive, forming stable amide bonds between cross-linkedmolecules within a pH range of about 7 to 9. In some cases,disuccinimidyl glutarate (DSG), a water-insoluble, homobifunctionalcrosslinker containing amine-reactive NHS esters at both ends, may beused for biconjugation. In some cases, N,N′-Disuccinimidyl carbonate(DSC), the smallest homobifunctional NHS ester crosslinking reagentavailable may be used. In some cases, Dimethyl adipimidate (DMA),Dimethyl pimelimidate (DMP), Dimethyl suberimidate (DMS), dimethyl3,3′-dithiobispropionimidate (DTBP),1,4-di-[3′-(2′-pyridyldithio)propionamido] butane, bismaleimidohexane,1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene,DFDNPS (4,4′-difluoro-3,3′-dinitrophenylsulfone),Bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde,Glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic dihydrazide,carbohydrazide, 3,3′-dimethylbenzidine, p-diaminodiphenyl, or haloacetylderivatives may be used as homobifunctional crosslinkers.

In some cases, heterobifunctional crosslinkers mediate the conjugationfor bioconjugation. Heterobifunctional reagents can be used to crosslinkproteins and other molecules in a two- or three-step process. In somecases, one protein may be modified with a heterobifunctional compoundusing the crosslinker's most reactive or most labile end. The modifiedprotein may then be purified from excess reagent by gel filtration orrapid dialysis. In some cases, heterobifunctionals contain at least onereactive group that displays extended stability in aqueous environments,therefore allowing purification of an activated intermediate beforeadding the second molecule to be conjugated. For instance, anN-hydroxysuccinimide (NHS ester-maleimide hetero-bifunctional can beused to react with the amine groups of one protein through its NHS esterend (the most labile functionality), while preserving the activity ofits maleimide functionality. Since the maleimide group has greaterstability in aqueous solution than the NHS ester group, amaleimide-activated intermediate may be created. After a quickpurification step, the maleimide end of the crosslinker can then be usedto conjugate to a sulfhydryl-containing molecule. Heterobifunctionalcrosslinking reagents may also be used to site-direct a conjugationreaction toward particular parts of target molecules. In some cases,amines may be coupled on one molecule while sulfhydryls or carbohydratesare targeted on another molecule. In some cases, heterobifunctionalreagents containing one photo-reactive end may be used to insertnonselectively into target molecules by UV irradiation. Anothercomponent of heterobifunctional reagents may be the cross-bridge orspacer that ties the two reactive ends together. Crosslinkers may beselected based not only on their reactivities, but also on the lengthand type of cross-bridge they possess. Some heterobifunctional familiesdiffer solely in the length of their spacer. The nature of thecross-bridge may also govern the overall hydrophilicity of the reagent.For instance, polyethylene glycol (PEG)-based cross-bridges createhydrophilic reagents that provide water solubility to the entireheterobifunctional compound. In some cases, a number ofheterobifunctionals contain cleavable groups within their cross-bridges,lending greater flexibility to the experimental design. A fewcrosslinkers contain peculiar cross-bridge constituents that actuallyaffect the reactivity of their functional groups. For instance, it isknown that a maleimide group that has an aromatic ring immediately nextto it is less stable to ring opening and loss of activity than amaleimide that has an aliphatic ring adjacent to it. In addition,conjugates destined for use in vivo may have different propertiesdepending on the type of spacer on the associated crosslinker. Somespacers may be immunogenic and cause specific antibody production tooccur against them. In other instances, the half-life of a conjugate invivo may be altered by the choice of cross-bridge, especially when usingcleavable reagents. In some cases, the heterobifunctional crosslinkermay be N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), standardSPDP, LC-SPDP, sulfo-LC-SPDP,succinimidyloxycarbonyl-α-methyl-α-(2-pyri-dyldithio) toluene,succinimidyl-4-(N-maleimidomethyl)cyclo-hexane-1-carboxylate,sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,m-maleimidobenzoyl-N-hydroxysuccinimide ester,m-maleimidobenzoyl-N-hydroxysulfo-succinimide ester,N-succinimidyl(4-iodoacetyl)aminobenzoate,sulfosuccinimidyl(4-iodoacetyl)amino-benzoate,succinimidyl-4-(p-maleimidophenyl)butyrate,N-(γ-maleimidobutyryloxy)succinimide ester,succinimidyl-3-(bromoacetamide)propionate, succinimidyl iodoacetate,4-(4-N-maleimidophenyl)butyric acid hydrazide,4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide,3-(2-pyridyldithio)propionyl hydrazide,N-hydroxysuccinimidyl-4-azidosalicylic acid,sulfosuccinimidyl-2-(p-azidosalicylamido) ethyl-1,3′-dithiopropionate,N-hydroxysulfosuccinimidyl-4-azido-benzoate,N-succinimidyl-6-(4′-azido-2′-nitropheny-lamino)hexanoate,sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate,N-5-Azido-2-nitrobenzoyloxysuccinimide,Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate,N-succinimidyl-(4-azidophenyl)1,3′-dithiopropionate, sulfosuccinimidyl4-(p-azidophenyl) butyrate, Sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate,sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate, p-Nitrophenyldiazopyruvate, p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate,1-(p-azidosalicylamido)-4-(iodoacetamido)butane,N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio) propionamide,Benzophenone-4-maleimide, p-azidobenzoyl hydrazide,4-(p-azidosalicylamido)butylamine, or p-azidophenyl glyoxal.

Other examples of crosslinkers, but not limited to, may beNHS-PEG₄-Azide, NHS-phosphine, N-γ-maleimidobutyryl-oxysulfosuccinimideester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl3-(2-pyridyldithio)propionate), sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, m-maleimidobenzoyl-N-hydroxysuccinimideester, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride,dimethyl pimelimidate, sulfosuccinimidyl6-(3′-(2-pyridyldithio)propionamido)hexanoate,6-(3′-[2-pyridyldithio]-propionamido)hexanoate,tris-(succinimidyl)aminotriacetate, Sulfo-NHS-LC-Diazirine,bismaleimidohexane, 1,4-bismaleimidobutane, sulfosuccinimidyl4-(N-maleimidophenyl)butyrate, Sulfo-SBED Biotin Label Transfer Reagent,succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate, succinimidyl3-(2-pyridyldithio)propionate, sulfosuccinimidyl6-(3′-(2-pyridyldithio)propionamido)hexanoate, L-Photo-Leucine,L-Photo-Methionine, sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, Pierce BS(PEG)5,sulfosuccinimidyl 2-((4,4′-azipentanamido)ethyl)-1,3′-dithiopropionate,Sulfo-NHS-SS-Diazirine, Pierce SM(PEG)n, NHS-dPEG-Mal,N-hydroxysulfosuccinimide, sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate,Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide Hydrochloride,N-α-maleimidoacet-oxysuccinimide ester, Sulfo-NHS-LC-Biotin,bis(sulfosuccinimidyl)suberate,trans-4-(maleimidylmethyl)cyclohexane-1-Carboxylate, bismaleimidohexane,1,8-bismaleimido-diethyleneglycol, N-β-maleimidopropionic acidhydrazide, N-succinimidyl 3-(2-pyridyldithio)-propionate,sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,3-(2-pyridyldithio)propionyl hydrazide, 4-(4-N-maleimidophenyl)butyricacid hydrazide, 3,3′-dithiobis(sulfosuccinimidyl propionate,bis(sulfosuccinimidyl) 2,2,4,4-glutarate-d4, orSuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

In some cases, the alkyne derivative attached to the solid support orSNAP may be, for example, dibenzocyclooctyne-amine,dibenzocyclooctyne-acid, dibenzocyclooctyne-N-hydroxysuccinimidyl ester,dibenzocyclooctyne-N-hydroxysuccinimidyl ester,dibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester,ibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester,Dibenzocyclooctyne-S—S—N-hydroxysuccinimidyl ester,dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester,dibenzocyclooctyne-PEG4-acid, dibenzocyclooctyne-maleimide,sulfo-dibenzocyclooctyne-biotin conjugate,(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate,(1R,8S,9s)-Bicyclo6.1.0non-4-yn-9-ylmethanol, APN-BCN,(1R,8S,9s)-Bicyclo6.1.0non-4-yn-9-ylmethanol, ethyl(1R,8S,9s)-bicyclo6.1.0non-4-ene-9-carboxylate, Alkyne-PEG5-acid,(R)-3-Amino-5-hexynoic acid hydrochloride, (S)-3-Amino-5-hexynoic acidhydrochloride, (R)-3-(Boc-amino)-5-hexynoic acid,(S)-3-(Boc-amino)-5-hexynoic acid, N-Boc pentyne-1-amine,4-pentyne-1-amine, Boc-propargyl-Gly-OH, 3-Ethynylaniline,4-Ethynylaniline, PC biotin-alkyne, Propargyl chloroformate,Propargyl-N-hydroxysuccinimidyl ester, N—Z-4-pentyne-1-amine,1-Azido-2-(2-(2-ethoxyethoxy)ethoxy)ethane,O-(2-Azidoethyl)heptaethylene glycol, Click-iT® DIBO-Alexa Fluor® 488,Click-iT® DIBO-Alexa Fluor® 555, Click-iT® DIBO-Alexa Fluor® 594,Click-iT® DIBO-Alexa Fluor® 647, Click-iT® DIBO TAMRA, Click-iT®DIBO-biotin, Click-iT® DIBO-amine, Click-iT® DIBO-maleimide, Click-iT®DIBO-succinimidyl ester, Alexa Fluor® 488 alkyne, Alexa Fluor® 555alkyne, triethylammonium salt, Alexa Fluor® 594 carboxamido-(5-(and6-)propargyl), bis(triethylammonium salt, 3-propargyloxypropanoic acid,succinimidyl ester, biotin alkyne, tetraacetyl fucose alkyne, OregonGreen® 488 alkyne*6-isomer*, iodoacetamide alkyne, or5-carboxytetramethylrhodamine propargylamide.

In some cases, the azide derivative attached to a solid support, SNAP,or biomolecule may be, for example, (S)-5-Azido-2-(Fmoc-amino)pentanoicacid, (S)-(−)-2-Azido-6-(Boc-amino)hexanoic acid (dicyclohexylammonium),(S)-2-Azido-3-(4-tert-butoxyphenyl)propionic acid cyclohexylammoniumsalt, L-Azidohomoalanine hydrochloride, (S)-2Azido-3-(3-indolyl)propionic acid cyclohexylammonium salt,(S)-2-Azido-3-methylbutyric acid cyclohexylammonium salt,(S)-2-Azido-3-phenylpropionic acid (dicyclohexylammonium) salt,Boc-3-azido-Ala-OH (dicyclohexylammonium) salt,N-Boc-4-azido-L-homoalanine (dicyclohexylammonium) salt,N-Boc-6-azido-L-norleucine (dicyclohexylammonium) salt,Boc-4-azido-Phe-OH, (S)-(−)-4-tert-Butyl hydrogen 2-azidosuccinate(dicyclohexylammonium) salt,N2-[(1,1-Dimethylethoxy)carbonyl]-N6-[(2-propynyloxy)carbonyl]-L-lysine,Fmoc-β-azido-Ala-OH, 2-Acetamido-2-deoxy-β-D-glucopyranosyl azide,2-Acetamido-2-deoxy-β-D-glucopyranosyl azide 3,4,6-triacetate,2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl azide,N-Azidoacetylgalactosamine-tetraacylated, N-Azidoacetylglucosamine,N-Azidoacetylglucosamine-tetraacylated,6-Azido-6-deoxy-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose,1-Azido-1-deoxy-β-D-galactopyranoside,1-Azido-1-deoxy-β-D-galactopyranoside tetraacetate,6-Azido-6-deoxy-D-galactose, 1-Azido-1-deoxy-β-D-glucopyranoside,2-Azido-2-deoxy-D-glucose, 6-Azido-6-deoxy-D-glucose,1-Azido-1-deoxy-β-D-lactopyranoside,3-Azido-2,3-dideoxy-1-O(tert-butyldimethylsilyl)-β-D-arabino-hexopyranose,2-Azido-D-galactose tetraacetate,1,2-Di-O-acetyl-3-azido-3-deoxy-5-O-(p-toluoyl)-D-ribofuranose,α-D-Mannopyranosyl azide tetraacetate,2,3,4,6-Tetra-O-acetyl-1-azido-1-deoxy-α-D-galactopyranosyl cyanide,2,3,4-Tri-O-acetyl-β-D-xylopyranosyl azide, 3′-Azido-3′-deoxythymidine,γ-(2-Azidoethyl)-ATP sodium salt solution, γ-[(6-Azidohexyl)-imido]-ATPsodium salt, (2'S)-2′-Deoxy-2′-fluoro-5-ethynyluridine,5-Ethynyl-2′-deoxycytidine, N6-Propargyl-ATP sodium salt,4-Acetamidobenzenesulfonyl azide,(E)-N-(2-Aminoethyl)-4-{2-[4-(3-azidopropoxy)phenyl]diazenyl}benzamidehydrochloride, Azidoacetic acid NHS ester, 1-Azidoadamantane,4-Azidoaniline hydrochloride,(4S)-4[(1R)-2-Azido-1-(benzyloxy)ethyl]-2,2-dimethyl-1,3-dioxolane,NHS-PEG₄-azide,[3aS-(3aα,4α,5β,7aα)]-5-Azido-7-bromo-3a,4,5,7a-tetrahydro-2,2-dimethyl-1,3-benzodioxol-4-ol,3′-Azido-3′-2-azido-1-methylquinolinium tetrafluoroborate,5-Azidopentanoic acid, 4-Azidophenacyl bromide, 4-Azidophenylisothiocyanate, 3-(4-Azidophenyl)propionic acid, 3-Azido-1-propanamine,3-Azido-1-propanol, Azo biotin-azide, Biotin picolyl azide, tert-Butyl2-(4-{[4-(3-azidopropoxy)phenyl]azo}benzamido)ethylcarbamate,4-Carboxybenzenesulfonazide, 7-(Diethylamino)coumarin-3-carbonyl azide,Ethidium bromide monoazide, Ethyl azidoacetate,4-Methoxybenzyloxycarbonyl azide, aryl azides, diazierines, orO-(2-Aminoethyl)-0′-(2-azidoethyl)heptaethylene glycol,bromoacetomido-PEG₃-azide, iodoacetamide-azide, Alexa Fluor® 488 azide,Alexa Fluor® 488 5-carboxamido-(6-azidohexanyl), bis(triethylammoniumsalt), Alexa Fluor® 555 azide triethylammonium salt, Alexa Fluor® 594carboxamido-(6-azidohexanyl), bis(triethylammonium salt), Alexa Fluor®647 azide triethylammonium salt, 3-(azidotetra(ethyleneoxy))propionicacid succinimidyl ester, biotin azide, L-azidohomoalanine,L-homopropargylglycine, Click-iT® farnesyl alcohol azide,15-azidopentadecanoic acid, 12-azidododecanoic acid, tetraacetylatedN-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine,tetraacetylated N-azidoacetylglucosamine, iodoacetamide azide, ortetramethylrhodamine 5-carboxamido-(6-azidohexanyl).

In some cases, the SNAPs may be covalently attached to the solid supportusing an inherent chemistry of the SNAP. In some cases, the solidsupport may be covered with functional groups that may be reactive tothe SNAP. These functional groups, for example, may be hydroxyl,carbonyl, carboxyl, amino, amides, azides, alkynes, alkenes, phosphates,sulfhydryl, thiols, isothiocyanates, isocyanates, acyl azides, NHSesters, silane, sulfonyl chlorides, aldehydes, esters, glyoxals,epoxides, oxiranes, alkanethiols, carbonates, aryl halides, imidoesters,carbodiimides, anhydrides, fluorophenyl esters, amines, thymines or acombination thereof. In some cases, the SNAP may have a functional groupthat may react with a functional group on the solid support to form acovalent bond. For example, a DNA SNAP may be attached to a solidsupport by reacting one or more thymines in the DNA with amines on thesolid support. For example, the —NH₂ at the N-terminus of a polypeptidechain or —COOH at the C-terminus of a polypeptide chain may react withan appropriate functional group and be attached to the solid supportthrough a covalent bond. In some cases, for example, the functionalgroup of a SNAP may be hydroxyl, carbonyl, carboxyl, amino, amides,azides, alkynes, silane, alkenes, phosphates, sulfhydryl, thiols,isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonylchlorides, aldehydes, esters, glyoxals, epoxides, oxiranes,alkanethiols, carbonates, aryl halides, imidoesters, carbodiimides,anhydrides, fluorophenyl esters, amines, thymines or a combinationthereof. Other bioconjugation processes, reactions, and functionalgroups are described elsewhere within that may be used to attach a SNAPto a solid support. Such a reaction could be spontaneous, or could beinduced by application of heat or ultraviolet radiation.

In some cases, silane chemistry may be employed for bioconjugation. Insome cases, functional silane compounds containing an organofunctionalor organo-reactive arm can be used to conjugate biomolecules toinorganic substrates. The appropriate selection of the functional orreactive group for a particular application can allow the attachment ofproteins, oligonucleotides, whole cells, organelles, or even tissuesections to substrates. The organosilanes used for these applicationsmay include functional or reactive groups such as hydroxyl, amino,aldehyde, epoxy, carboxylate, thiol, and even alkyl groups to bindmolecules through hydrophobic interactions. In some cases,3-Aminopropyltriethoxysilane (APTES) and 3-Aminopropyltrimethoxysilaneare used to create a functional group on an inorganic surface orparticle. In some cases, once deposited on a substrate, the alkoxygroups form a covalent polymer coating with the primary amine groupssticking off the surface and available for subsequent conjugation.Carboxyl- or aldehyde-containing ligands may be directly coupled to theaminopropyl groups using a carbodiimide reaction or reductive amination.In some cases, alternatively, surfaces initially derivatized with anaminopropylsilane compound can be modified further with spacer arms orcrosslinkers to create reactive groups for coupling affinity ligands orbiomolecules. For instance, the amine groups may be derivatized with anNHS-PEGn-azide compound for use in click chemistry or Staudingerligation reactions for linking proteins or other biomolecules. In somecases, APTES-modified surfaces may be further derivatized withamine-reactive crosslinkers to create additional surface characteristicsand reactivity. Modification with NHS-PEG4-azide forms a hydrophilic PEGspacer terminating in an azido group that can be used in a clickchemistry or Staudinger ligation reaction to couple other molecules.

In some cases, other crosslinking agents that contain an amine-reactivegroup on one end also may be used to modify and activate theAPTES-modified substrate. Surfaces may be designed to contain, forinstance, reactive hydrazine or aminooxy groups for conjugation withcarbonyl-containing molecules, such as aldehydes formed throughperiodate oxidation of carbohydrates or natively present at the reducingend of sugars and glycans. In other instances, crosslinking reagents maycontain an amine-reactive group on one end to attach to theAPTES-modified substrate and the other end can be a moiety that canintercalate DNA bases (for example, NHS esters of psoralen or otherintercalating agents). Once SNAPs are immobilized by the intercalatinginteraction, they can be covalently crosslinked by thymidine adducts byexposure to UV light.

In some cases, the amine groups on ATPS surfaces may be acylated usingglutaric anhydride to create carboxylate functionalities, which werethen activated with NHS/DCC to form the NHS ester. This derivative couldbe used to couple amine-containing proteins and other molecules viaamide bond formation. In a second activation strategy, the aminopropylgroups on the surface were activated with 1,4-phenylenediisothiocyanate(PDITC) to create terminal isothiocyanate groups for coupling amines.Both methods resulted in the successful coupling of amine-dendrimers tosilica surfaces for use in arrays. In some cases, amine surfacesprepared using an aminosilane compound can be modified to containcarboxylate groups using the following protocol involving the reactionwith an anhydride, such as succinic anhydride or glutaric anhydride.After modification, the carboxylates then can be used to coupleamine-containing molecules using a carbodiimide reaction with EDC plussulfo-NHS. In some cases, modification of an APTES surface with glutaricanhydride creates terminal carboxylates for coupling of amine-containingligands which may be used for bioconjugation.

In some cases, aminosilane surfaces also may be activated by use of abifunctional crosslinker to contain reactive groups for subsequentcoupling to biomolecules. In one such reaction, N,N′-disuccinimidylcarbonate (DSC) was used to react with the amines on a slide surface andcreate terminal NHS-carbonate groups, which then could be coupled toamine-containing molecules, which may be used for bioconjugation. Insome cases, APTES-modified surfaces can be activated with DSC to formamine-reactive succinimidyl carbonates for coupling proteins or otheramine-containing molecules.

In some cases, silane coupling agents containing carboxylate groups maybe used to functionalize a surface with carboxylic acids for subsequentconjugation with amine-containing molecules. For example,carboxyethylsilanetriol contains an acetate organo group on asilanetriol inorganic reactive end. The silanetriol component may bereactive immediately with inorganic —OH substrates without priorhydrolysis of alkoxy groups, as in the case with most other silanizationreagents. In some cases, carboxyethylsilanetriol has been used to addcarboxylate groups to fluorescent silica nanoparticles to coupleantibodies for multiplexed bacteria monitoring. This reagent can be usedin similar fashion to add carboxylate functionality to many inorganic ormetallic nano-materials, which also will create negative chargerepulsion to maintain particle dispersion in aqueous solutions. In somecases, covalent coupling to the carboxylated surface then can be done byactivation of the carboxylic acid groups with a carbodiimide tofacilitate direct reaction with amine-containing molecules or to formintermediate NHS esters, which may be used for bioconjugation. In somecases, carboxylethylsilanetriol can be used to modify an inorganicsubstrate to containing carboxylate groups for coupling amine-containingligands.

In some cases, silane modification agents such as glycidoxy compoundsmay be utilized for bioconjugation to a surface substrate. Glycidoxycompounds contain reactive epoxy groups. Surfaces covalently coated withthese silane coupling agents can be used to conjugate thiol-, amine-, orhydroxyl-containing ligands, depending on the pH of the reaction. Insome cases, 3-glycidoxy-propyltrimethoxysilane (GOPTS) or3-glycidoxypro-pyltriethoxysilane can be used to link inorganic silicaor other metallic surfaces containing —OH groups with biologicalmolecules containing any three of these major functional groups. In somecases, epoxy-containing silane coupling agents form reactive surfacesthat can be used to couple amine-, thiol-, or hydroxyl-containingligands which may be used for bioconjugation.

In some cases, the reaction of the epoxide with a thiol group yields athioether linkage, whereas reaction with a hydroxyl gives an ether andreaction with an amine results in a secondary amine bond. The relativereactivity of an epoxy group is thiol>amine>hydroxyl, and this may bereflected by the optimal pH range for each reaction. In this case, thelower the reactivity of the functional group the higher the pH requiredto drive the reaction efficiently.

In some cases, isocyanates groups may be utilized for bioconjugation toa surface support. Isocyanate groups are extremely reactive towardnucleophiles and will hydrolyze rapidly in aqueous solution which areespecially useful for covalent coupling to hydroxyl groups undernonaqueous conditions, which may be appropriate for conjugation to manycarbohydrate ligands. Silanization can be accomplished in dry organicsolvent to form reactive surfaces while preserving the activity of theisocyanates. Isocyanatopropyltriethoxysilane (ICPTES) contains anisocyanate group at the end of a short propyl spacer, which may beconnected to the triethoxysilane group useful for attachment toinorganic substrates. In some cases, the isocyanate-containing silanecoupling agent can be used to couple hydroxyl-containing molecules toinorganic surfaces which may be used for bioconjugation.

In some cases, ICPTES may be used to create novel chitosan-siloxanehybrid polymers by coupling the isocyanate groups to the functionalgroups of the carbohydrate and forming a silica polymer using thetriethoxysilane backbone. In some cases, ICPTES and APTES have been usedin combination to create organically modified silica xerogels throughcarboxylic acid solvolysis that formed hybrid materials with luminescentproperties.

In some cases, nanoparticles or microparticles may be utilized as asurface support for bioconjugation. In some cases, particle types andcompositions of almost limitless shape and size, including spherical,amorphous, or aggregate particles, as well as elaborate geometric shapeslike rods, tubes, cubes, triangles, and cones. In addition, newsymmetrical organic constructs have emerged in the nanometer range thatinclude fullerenes (e.g., Bucky-balls), carbon nanotubes, anddendrimers, which are highly defined synthetic structures used asbioconjugation scaffolds. The chemical composition of particles may bejust as varied as their shape. Particles can comprise of polymers orcopolymers, inorganic constructs, metals, semiconductors,superparamagnetic composites, biodegradable constructs, syntheticdendrimers, and dendrons. Polymeric particles can be constructed from anumber of different monomers or copolymer combinations. Some of the morecommon ones include polystyrene (traditional “latex” particles),poly(styrene/divinylbenzene) copolymers, poly(styrene/acrylate)copolymers, polymethylmethacrylate (PMMA), poly (hydroxyethylmethacrylate) (pHEMA), poly (vinyltoluene), poly(styrene/butadiene)copolymers, and poly(styrene/vinyltoluene) copolymers. In some cases, bymixing into the polymerization reaction combinations of functionalmonomers, one can create reactive or functional groups on the particlesurface for subsequent coupling to affinity ligands. One example of thismay be a poly(styrene/acrylate) copolymer particle, which createscarboxylate groups within the polymer structure, the number of which maybe dependent on the ratio of monomers used in the polymerizationprocess. In some cases, inorganic particles are used extensively invarious bioapplications. For example, gold nanoparticles may be used fordetection labels for immunohistochemical (IHC) staining and lateral flowdiagnostic testing. In some cases, the use of particles inbioapplications like bioconjugation involves the attachment of affinitycapture ligands to their surface, by either passive adsorption orcovalent coupling. The coupling of an affinity ligand to such particlescreates the ability to bind selectively biological targets in complexsample mixtures. The affinity particle complexes can thus be used toseparate and isolate proteins or other biomolecules or to specificallydetect the presence of these targets in cells, tissue sections, lysates,or other complex biological samples. In some cases, the reactions usedfor coupling affinity ligands to nanoparticles or microparticles arebasically the same as those used for bioconjugation of moleculesdescribed herein.

In some cases, particle type used for bioapplications (e.g.bioconjugation) may be the polymeric microsphere or nano-sphere, whichcomprises a spherical, nonporous, “hard” particle made up of long,entwined linear or crosslinked polymers. In some cases, creation ofthese particles involves an emulsion polymerization process that usesvinyl monomers, sometimes in the presence of divinyl crosslinkingmonomers. In some cases, larger microparticles may be built fromsuccessive polymerization steps through growth of much smallernanoparticle seeds. In some cases, polymeric particles comprise ofpolystyrene or copolymers of styrene, like styrene/divinylbenzene,styrene/butadiene, sty-rene/acrylate, or styrene/vinyltoluene. Othercommon polymer supports include polymethylmethacrylate (PMMA),polyvinyltoluene, poly(hydroxyethyl meth-acrylate) (pHEMA), and thecopolymer poly(ethylene glycol dimethacrylate/2-hydroxyethylmetacrylate)[poly(EGDMA/HEMA)].

In some cases, one method of attaching biomolecules to hydrophobicpolymeric particles may be the use of passive adsorption. In some cases,protein adsorption onto hydrophobic particles takes place through stronginteractions of nonpolar or aromatic amino acid residues with thesurface polymer chains on the particles with concomitant exclusion ofwater molecules. Since proteins usually contain hydrophobic corestructures with predominately hydrophilic surfaces, their interactionwith hydrophobic particles must involve significant conformationalchanges to create large-scale hydrophobic contacts.

In some cases, particle types contain functional groups that are builtinto the polymer backbone and displayed on their surface. The quantityof these groups can vary widely depending on the type and ratios ofmonomers used in the polymerization process or the degree of secondarysurface modifications that have been performed. In some cases,functionalized particles can be used to couple covalently biomoleculesthrough the appropriate reaction conditions.

Common Functional Groups or Reactive Groups on Particles forBioconjugation

In some cases, a particle may couple with a crosslinker forbioconjugation.

In some cases, the rate of attachment of DNA SNAPs s to the solidsupport, or the efficacy or strength of attachment, may be altered byaltering the sequence of DNA comprising the SNAP. For example, in thecase of a DNA SNAP attached to a solid support by a reaction involvingone or more thymines the attachment may be varied by varying the numberof thymines in the DNA sequence. In some cases, increasing the number ofthymines may facilitate the attachment of the SNAP to the solid support.

In some cases, the solid support may be a part of a flow cell. In somecases, the SNAPs may be attached to a solid support in a flow cell. Insome cases, the SNAPs may be directly conjugated to a solid support in aflow cell. In some cases, the SNAPs may be adsorbed to a solid supportin a flow cell. Attaching the SNAPs in the flow cell may allowvisualization of the SNAPs as they attach to the solid support. Theattachment of the SNAPs may be optimized by monitoring the number ofattached SNAPs compared to the number of attachment sites during theattachment process. In some cases, the attachment of the SNAPs may beoptimized by monitoring the area of the solid support covered by theSNAPs and the area of the solid support that may be unoccupied by theSNAPs during the attachment process.

In some cases, the SNAPs may be conjugated directly in a flow cell. Insome cases, the SNAPs may be conjugated to a surface within the flowcell. In some cases, the SNAPs may be conjugated to a surface within theflow cell before being conjugated to the biological, chemical, orphysical entities. In some cases, a biological, chemical, or physicalentity may be flowed into a flow cell and conjugated to a SNAP that maybe already conjugated to the solid support. In some cases, a biological,chemical, or physical entity may be conjugated to a SNAP before saidSNAP may be introduced into a flow cell and conjugated to a solidsupport in a flow cell. In some cases, a biological, chemical, orphysical entity and a SNAP may be introduced into a flow cell andconjugated to each other within the flow cell, before the SNAP may beconjugated to a solid support within the flow cell.

In some cases, the biological, chemical, or physical entities may beconjugated to the SNAPs prior to attaching the SNAPs to a solid support.After performing such a reaction the products may be purified toseparate out conjugated SNAP-biological/chemical entity moieties fromunconjugated SNAPs and biological/chemical entities.

The methods of this disclosure may be used to spatially separatebiological, chemical, or physical entities. In some embodiments, methodsof this disclosure may be used to spatially separate proteins, smallmolecules, DNAs, RNAs, glycoproteins, metabolites, carbohydrates,enzymes, or antibodies. In some embodiments, methods of this disclosuremay be used to spatially separate complexes, such as protein complexescomprising two or more proteins, protein nucleic acid complexes, orother complexes. In some cases, the methods may be used to spatiallyseparate viral particles or viroids. In some cases, the methods may beused to separate cells, such as bacterial cells, microbial cells,mammalian cells or other cells.

In some embodiments, the SNAP may be formed on the seed prior to theseed being attached to the biological, chemical, or physical entity.

In some embodiments this disclosure provides a composition comprising anucleic acid SNAP attached to a protein, a nucleic acid SNAP attached toa small molecule, a nucleic acid SNAP attached to a protein complex, anucleic acid SNAP attached to a protein nucleic acid SNAP, a nucleicacid SNAP attached to a carbohydrate, a nucleic acid SNAP attached to aviral particle or a nucleic acid SNAP attached to a cell.

In some embodiments this disclosure provides a composition comprising adendrimer attached to a protein, a dendrimer attached to a smallmolecule, a dendrimer attached to a protein complex, a dendrimerattached to a protein dendrimer, a dendrimer attached to a carbohydrate,a dendrimer attached to a viral particle or a dendrimer attached to acell.

In some cases, the biological, chemical, or physical entities may beeluted from the solid support either by cleaving a photo-cleavable bond,or by chemically or enzymatically digesting the SNAP.

In some cases, the biological, chemical, or physical entities may attachto the solid support directly, while the SNAPs occlude other biological,chemical, or physical entities from attaching in the immediate vicinity.In some cases the biological, chemical, or physical entities may attachdirectly to an attachment site within a microwell or nanowell, and thesize of the SNAPs may be selected to prevent more than one SNAP fromoccupying the microwell or nanowell. In such cases, the SNAP may beremoved, either by cleaving a photo-cleavable bond, or by chemically orenzymatically digesting the SNAP.

In some embodiments, SNAPs of this disclosure may be used asnanoparticles. For example, SNAPs of this disclosure may be used asnanoparticles for detection or visualization. In some cases, a nucleicacid SNAP may be formed which incorporates modified nucleotides whichcomprise fluorescent moieties. Any fluorescently labeled nucleotideknown in the art may be used in a SNAP of this disclosure. Examples offluorescently labeled nucleotides include, but are not limited to, AlexaFluor™ 555-aha-dCTP, Alexa Fluor™ 555-aha-dUTP, 1 mM in TE buffer, AlexaFluor™ 647 ATP (Adenosine 5′-Triphosphate, Alexa Fluor™ 6472′-(or-3′)-O—(N-(2-Aminoethyl) Urethane), Hexa(Triethylammonium) Salt),Alexa Fluor™ 647-aha-dCTP, Alexa Fluor™ 647-aha-dUTP, 1 mM in TE buffer,BODIPY™ FL ATP (Adenosine 5′-Triphosphate, BODIPY™ FL2′-(or-3′)-O—(N-(2-Aminoethyl)Urethane), Trisodium Salt), 5 mM inbuffer, BODIPY™ FL ATP-γ-S, Thioester (Adenosine5′-O-(3-Thiotriphosphate), BODIPY™ FL Thioester, Sodium Salt), BODIPY™FL GDP (Guanosine 5′-Diphosphate, BODIPY™ FL2′-(or-3′)-O—(N-(2-Aminoethyl) Urethane), Bis (Triethylammonium) Salt),ChromaTide™ Alexa Fluor™ 488-5-UTP, ChromaTide™ Alexa Fluor™ 488-5-dUTP,ChromaTide™ Alexa Fluor™ 546-14-UTP, ChromaTide™ Alexa Fluor™546-14-dUTP, ChromaTide™ Alexa Fluor™ 568-5-dUTP, ChromaTide™ AlexaFluor™ 594-5-dUTP, ChromaTide™ Fluorescein-12-dUTP, ChromaTide™ TexasRed™-12-dUTP, Fluorescein-12-dUTP Solution (1 mM),Fluorescein-aha-dUTP-1 mM in TE Buffer, Guanosine5′-O-(3-Thiotriphosphate), BODIPY™ FL Thioester, Sodium Salt (BODIPY™ FLGTP-γ-S, Thioester), Guanosine 5′-Triphosphate, BODIPY™ FL2′-(or-3′)-O—(N-(2-Aminoethyl) Urethane), Trisodium Salt (BODIPY™ FLGTP), Guanosine 5′-Triphosphate, BODIPY™ TR2′-(or-3′)-O—(N-(2-Aminoethyl) Urethane), Trisodium Salt (BODIPY™ TRGTP), MANT-ADP (2′-(or-3′)-O—(N-Methylanthraniloyl) Adenosine5′-Diphosphate, Disodium Salt), MANT-ATP(2′-(or-3′)-O—(N-Methylanthraniloyl) Adenosine 5′-Triphosphate,Trisodium Salt), MANT-GDP (2′-(or-3′)-O—(N-Methylanthraniloyl) Guanosine5′-Diphosphate, Disodium Salt), MANT-GMPPNP(2′-(or-3′)-O—(N-Methylanthraniloyl)-β:γ-Imidoguanosine 5′-Triphosphate,and Trisodium Salt), MANT-GTP (2′-(or-3′)-O—(N-Methylanthraniloyl)Guanosine 5′-Triphosphate, Trisodium Salt).

In some cases, a SNAP of this disclosure may be designed such thatprobes may be attached onto the surface of the SNAP. A SNAP withattached probes may be used as a detection reagent. In some cases, aSNAP with attached probes may be also labeled with fluorescent moietiesto form a fluorescent detection reagent. In some cases, a SNAP withattached probes and fluorescent moieties may provide a high degree ofsignal amplification. The amount of probes on the SNAP may be titratedto achieve a desired degree of sample amplification. In some cases,differently sized SNAPs may be attached to different probes. In somecases, differently colored SNAPs may be attached to different probes. Insome cases a library of different probes may be attached tofluorescently labeled SNAPs such that a first probe may be attached to aSNAP which may be a different size and/or color from a SNAP each otherprobe may be attached to.

A biomolecule may be coupled to an array on a solid support that iscoupled to a light sensing device comprising a plurality of pixels,where each pixel is capable of independently sensing incident light. Thebiomolecule may be directly coupled to the support or may be coupled bylinking molecule (e.g., a SNAP or a magnetic nanoparticle). Thebiomolecule may be coupled to the solid support coupled to the lightsensing device by covalent conjugation or a non-covalent interaction(e.g., electrostatic adhesion). Coupled biomolecules on an array maycomprise a protein, peptide, DNA molecule, RNA molecule, carbohydrate,binding ligand, or a complex comprising more than one molecule of any ofthe aforementioned biomolecules.

An array comprising a plurality of biomolecules may have anyconfiguration of biomolecules on the array. An array comprising aplurality of biomolecules may comprise about 10, 100, 1000, 10000,100000, 1000000, 10000000, 100000000, 1000000000, 10000000000 or morethan 10000000000 biomolecules. An array comprising a plurality ofbiomolecules may comprise at least about 10, 100, 1000, 10000, 100000,1000000, 10000000, 100000000, 1000000000, 10000000000 or more than10000000000 biomolecules. An array comprising a plurality ofbiomolecules may comprise about 10000000000, 1000000000, 100000000,10000000, 1000000, 100000, 10000, 1000, 100, 10 or less than 10biomolecules.

An array comprising a plurality of biomolecules may comprise an orderedarray. An ordered array comprising a plurality of biomolecules may becreated on a structured or ordered surface (e.g., an array created bynanolithography of a substrate). An array comprising a plurality ofbiomolecules may comprise a non-ordered array (e.g., random distributionof attachment sites for biomolecules). An array comprising a pluralityof biomolecules may have a one-dimensional configuration,two-dimensional configuration (attachment to a flat surface or a planarsurface), or three-dimensional configuration (attachment on a non-planaror curved surface, e.g., nanowells, beads).

An array comprising a plurality of coupled biomolecules may havecomplete occupancy of attachment sites or less than complete occupancyof binding sites. An array comprising a plurality of binding sites maycomprise a sparse array if it has more unoccupied attachments sites thanoccupied attachment sites. An array comprising a plurality of bindingsites may comprise a dense array if it has more occupied attachmentsites than unoccupied sites. An array may be characterized by anoccupancy count or a vacancy count. An occupancy count or a vacancycount may be absolute (e.g., M attachment sites are unoccupied, Nattachment sites are occupied) or relative (e.g., X % of binding sitesare unoccupied, Y % of attachment sites are occupied). An arraycomprising a plurality of biomolecules may have an occupancy count orvacancy count of about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, ormore than 99.9%. An array comprising a plurality of biomolecules mayhave an occupancy count or vacancy count of at least about 0.1%, 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 99%, 99.9%, or more than 99.9%. An array comprisinga plurality of biomolecules may have an occupancy count or vacancy countof no more than about 99.9%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.1%, orless than 0.1%. An occupancy count or a vacancy count may be determinedover an entire array or a subsection, area, or region of an arraycomprising a plurality of biomolecules.

An array may be characterized by an occupancy rate or a vacancy rate. Anoccupancy rate or vacancy rate may be defined as the number of occupiedor unoccupied attachment sites, respectively, per some reference numberof sites. An array may have an occupancy rate or a vacancy rate of about1 in 10, 1 in 20, 1 in 50, 1 in 100, 1 in 200, 1 in 500, 1 in 1000, 1 in2000, 1 in 5000, 1 in 10000, 1 in 20000, 1 in 50000, 1 in 100000, 1 in200000, 1 in 500000, 1 in 1000000, or more than 1 in 1000000. An arraymay have an occupancy rate or a vacancy rate of at least about 1 in 10,1 in 20, 1 in 50, 1 in 100, 1 in 200, 1 in 500, 1 in 1000, 1 in 2000, 1in 5000, 1 in 10000, 1 in 20000, 1 in 50000, 1 in 100000, 1 in 200000, 1in 500000, 1 in 1000000, or more than 1 in 1000000. An array may have anoccupancy rate or a vacancy rate of no more than about 1 in 1000000, 1in 500000, 1 in 200000, 1 in 100000, 1 in 50000, 1 in 20000, 1 in 10000,1 in 5000, 1 in 2000, 1 in 1000, 1 in 500, 1 in 200, 1 in 100, 1 in 50,1 in 20, 1 in 10, or less than 1 in 10. An occupancy rate or a vacancyrate may be determined over an entire array or a subsection, area, orregion of an array comprising a plurality of biomolecules.

A plurality of biomolecules may be coupled to a solid support at asurface density that is determined by the size of each pixel of a lightsensing device. Biomolecules may be conjugated to a solid support suchthat each pixel of the light sensing device detects a singlebiomolecule. In some cases, biomolecules may be coupled to a solidsupport such that each biomolecule is detected by more than one pixel.FIGS. 9A-9C depict top-down views of pixel arrays with biomoleculesdeposited at differing densities. FIG. 9A depicts a view of a pixelarray comprising 16 contiguous pixels 910 with biomolecules 920deposited at a density of about 1 biomolecule per pixel, although theoccupancy of the array is less than 100%. FIGS. 9B and 9C depict arrayswith 2 pixels 910 per biomolecule 920 and 4 pixels 910 per biomolecule920, respectively, at less than 100% occupancy of binding sites forbiomolecules. Having more than one pixel available per biomolecule mayincrease the accuracy and sensitivity of the detection device.Biomolecules may be coupled to the solid support that is coupled to thelight sensing device at a surface density of at least about 1 pixel perbiomolecule, 2 pixels per biomolecule, 3 pixels per biomolecule, 4pixels per biomolecule, 5 pixels per biomolecule, 6 pixels perbiomolecule, 7 pixels per biomolecule, 8 pixels per biomolecule, 9pixels per biomolecule, 10 pixels per biomolecule, 15 pixels perbiomolecule, 20 pixels per biomolecule, 25 pixels per biomolecule, 50pixels per biomolecule, 100 pixels per biomolecule, or more than about100 pixels per biomolecule. Biomolecules may be coupled to the solidsupport that is coupled to the light sensing device at a surface densityof no more than about 100 pixel per biomolecule, 50 pixels perbiomolecule, 25 pixels per biomolecule, 20 pixels per biomolecule, 15pixels per biomolecule, 10 pixels per biomolecule, 9 pixels perbiomolecule, 8 pixels per biomolecule, 7 pixels per biomolecule, 6pixels per biomolecule, 5 pixels per biomolecule, 4 pixels perbiomolecule, 3 pixels per biomolecule, 2 pixels per biomolecule, or lessthan 2 pixels per biomolecule. A solid support may have a biomoleculeoccupancy based upon the total number of available attachment sites ofat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more than 99%. A solidsupport may have a biomolecule occupancy based upon the total number ofavailable attachment sites of no more than about 99%, 95%, 90%, 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, 5%, or less than 5%.

Each pixel of a light sensing device coupled to a solid support may becapable of detecting the interaction of incident light with abiomolecule coupled to the solid support. In some cases, the interactionof the incident light with the biomolecule may comprise the absorbanceof the incident light, the transmission of the incident light, thereflection or refraction of the incident light, or the emission ofphotons of differing wavelength in response to incident light. Eachpixel of the light sensing device may detect the presence or absence ofincident photons. Each pixel of the light sensing device may be capableof detecting the intensity of incident photons.

In some cases, a light sensing device coupled to a solid support maydetect the interaction of an affinity reagent or a plurality of affinityreagents with one or more biomolecules coupled to the solid support. Theinteraction of the affinity reagent may include a non-covalentinteraction such as binding or complex formation of the affinity reagentwith the biomolecule. The interaction of the affinity reagent mayinclude a covalent interaction such as the reaction of the affinityreagent with the biomolecule. A biomolecule may comprise a detectablelabel such as a fluorophore, radiolabel, or bioluminescent moiety thatmay transmit light to a pixel of a light sensing device.

FIG. 10 depicts a cross-sectional view of a light sensing device coupledto an array of biomolecules, in accordance with some embodiments. Asolid support 1010 is coupled to an array of light sensing pixels, witheach pixel comprising a nanowell 1020 and a light sensing element 1030.At the surface of the solid support 1010, linking molecules 1040 (e.g.SNAPs) bind differing biomolecules 1051, 1052, and 1053 to the solidsupport. Detectable affinity reagents 1060 bind to biomolecules 1051 and1053, but do not bind to biomolecule 1052. In the presence of anexciting radiation field, photons of emitted light may be transferredfrom the detectable labels 1060 to the corresponding light sensingelement 1030.

Affinity reagents may include aptamers, antibodies, antibody fragments,mini-peptide binders, avimers, peptimers, phage display, metalnanoparticles (e.g., TiO2 nanoparticles), and magnetic nanoparticles(e.g., paramagnetic nanoparticles). Affinity reagents may also includereagents that interact in identifiable manners with biomolecules, suchas kinases, proteases, restriction enzymes, reducing agents, oxidizingagents, and reactive labels (e.g., amine-reactive dyes or thiol-reactivedyes). An interaction of a reactive affinity reagent may be detecteddirectly by methods such as fluorescence detection, luminescencedetection, or surface plasmon resonance. An interaction of a reactiveaffinity reagent may be detected by a change in the properties of abiomolecule (e.g., removal of a protein-linked fluorophore caused by theaction of a protease). An interaction of a reactive affinity reagent maybe detected indirectly by secondary interactions with reactive affinityreagents. For example, an affinity reagent may comprise a cross-linkingreagent that contains an amine-reactive or thiol-reactive group forreacting with a protein side chain and a second reactive group that canbe reacted with other detectable reagents (fluorophores, magneticparticles, etc.). In some cases, more than one affinity reagent may belinked to form an affinity reagent complex (e.g., two linked aptamers,an aptamer linked to a protease). Affinity reagents complexes may beused to obtain additional information when characterizing theinteraction of the affinity reagent complex with a protein or peptide.For example, an affinity reagent complex may be used to determine if anepitope is within a certain proximity to another epitope.

An affinity agent may have a known or characterized degree ofnonspecificity. A degree of nonspecificity may be defined as theproperty of binding to more than one unique biomolecule. For example, anaffinity reagent may comprise a degree of nonspecificity if it binds toany protein that contains an epitope from the family of epitopes definedby the sequence X1-X2-G, where X1 and X2 may be any amino acid residueor a known subset of amino acid residues (e.g., M-F-G, K-W-G, S-K-G,W-A-G, etc.). An affinity reagent with a degree of nonspecificity maybind to more than one family of epitopes (e.g., A-X2-T and G-X2-E). Anaffinity reagent with a degree of nonspecificity may bind to epitopeswith a common structural pattern (e.g., all positive or neutral chargeside chains). An affinity reagent with a degree of nonspecificity maybind to a random, known set of amino acid epitopes (e.g., K-D-S, R-M-W,D-T-C, etc.). An affinity reagent may have a degree of nonspecificity ifit binds to more than one protein molecule with a family of proteinsmolecules or if it binds to more than one type of protein molecule. Anaffinity reagent may have a known or characterized degree ofnonspecificity if the binding of the affinity reagent has been observedto bind to more than one protein molecule. In some cases, the degree ofnonspecificity of an affinity reagent may be described in aprobabilistic fashion (e.g., binding of an affinity reagent is observedin at least 25%, 50%, 75%, 90% or more of proteins comprising theepitope G-X-G). An affinity reagent with a degree of nonspecificity maybe observed to bind to a subset of protein molecules within a proteomewith the binding being linked to a known common structural elementwithin the subset of protein molecules (e.g., a common epitope, a commonpost-translational modification). An affinity reagent with a degree ofnonspecificity may be observed to bind to a subset of protein moleculeswithin a proteome without the binding being linked to a known commonstructural element within the subset of protein molecules (e.g., nocommon epitope, no common post-translational modification). Each uniqueaffinity reagent may have a unique binding profile when interacting witha plurality of differing biomolecules (e.g., a heterogeneous pluralityof proteins derived from a single cell) such that the affinity reagentmay only be detected at pixels where a biomolecule containing a targetepitope is located. A series or sequence of differing affinity reagentsmay produce a series or sequence of detected binding patterns whenallowed to interact with a plurality of biomolecules coupled to thesolid support or a light sensing device.

The interaction of an affinity reagent with a biomolecule may occur overa particular time interval. The interaction of an affinity reagent mayoccur for a long enough time interval for the interaction to be detectedby the light sensing device. A pixel of a light sensing device may becapable of detecting an interaction between an affinity reagent and abiomolecule that occurs for at least about 1 microsecond (μs), 10 μs, 50μs, 100 μs, 250 μs, 500 μs, 1 millisecond (ms), 10 ms, 50 ms, 100 ms,250 ms, 500 ms, 1 second (s), 5 s, 10 s, 30 s, 1 minute (min), 5 min, 10min, or more than about 10 minutes. A pixel of a light sensing devicemay be capable of detecting an interaction between an affinity reagentand a biomolecule that occurs for no more than about 10 min, 5 min, 1min, 30 second (s), 10 s, 5 s, 1 s, 500 millisecond ms, 250 ms, 100 ms,50 ms, 10 ms, 1 ms, 500 μs, 250 μs, 1000 μs, 50 μs, 10 μs, 1 μs, or lessthan about 1 μs.

A pixel on a light sensing device may be configured to minimizecross-talk or incident light emitted from entities not associated withthe pixel. In some cases, adjacent pixels on a light sensing device maybe spaced, oriented or shaped to minimize cross-talk or incident lightemitted from entities not associated with the pixel. For example, aplurality of biomolecules (e.g., proteins) may be coupled to a solidsupport such that each individual biomolecule is coupled to a portion ofthe solid support associated with one or more unique pixels on a lightsensing device. The biomolecules may have light-emitting orlight-absorbing entities (e.g., fluorophores) that interact with thebiomolecules (e.g., an affinity reagent) at some or all of thebiomolecules. A pixel may be arranged to minimize the amount of lightreceived from a light-emitting entity not associated with the pixel. Insome cases, the amount of incident light at a particular pixel may fallbelow the threshold of detection for the pixel. In some cases, theincident light at a particular pixel may comprise no more than about50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.9%,0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.005%,0.001%, or less than 0.001% light from non-associated entities.

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 3 shows a computer system 301that is programmed or otherwise configured to, for example, acquirepixel information of an array of biological, chemical, or physicalentities; and detect the array of biological, chemical, or physicalentities based at least in part on the acquired pixel information. Thecomputer system 301 can regulate various aspects of analysis,calculation, and generation of the present disclosure, such as, forexample, acquiring pixel information of an array of biological,chemical, or physical entities; and detecting the array of biological,chemical, or physical entities based at least in part on the acquiredpixel information. The computer system 301 can be an electronic deviceof a user or a computer system that is remotely located with respect tothe electronic device. The electronic device can be a mobile electronicdevice.

The computer system 301 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 305, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 301 also includes memory or memorylocation 310 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 315 (e.g., hard disk), communicationinterface 320 (e.g., network adapter) for communicating with one or moreother systems, and peripheral devices 325, such as cache, other memory,data storage and/or electronic display adapters. The memory 310, storageunit 315, interface 320 and peripheral devices 325 are in communicationwith the CPU 305 through a communication bus (solid lines), such as amotherboard. The storage unit 315 can be a data storage unit (or datarepository) for storing data. The computer system 301 can be operativelycoupled to a computer network (“network”) 330 with the aid of thecommunication interface 320. The network 330 can be the Internet, aninternet and/or extranet, or an intranet and/or extranet that is incommunication with the Internet. The network 330 in some cases is atelecommunication and/or data network. The network 330 can include oneor more computer servers, which can enable distributed computing, suchas cloud computing. For example, one or more computer servers may enablecloud computing over the network 330 (“the cloud”) to perform variousaspects of analysis, calculation, and generation of the presentdisclosure, such as, for example, acquiring pixel information of anarray of biological, chemical, or physical entities; and detecting thearray of biological, chemical, or physical entities based at least inpart on the acquired pixel information. Such cloud computing may beprovided by cloud computing platforms such as, for example, Amazon WebServices (AWS), Microsoft Azure, Google Cloud Platform, and IBM cloud.The network 330, in some cases with the aid of the computer system 301,can implement a peer-to-peer network, which may enable devices coupledto the computer system 301 to behave as a client or a server.

The CPU 305 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 310. The instructionscan be directed to the CPU 305, which can subsequently program orotherwise configure the CPU 305 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 305 can includefetch, decode, execute, and writeback.

The CPU 305 can be part of a circuit, such as an integrated circuit. Oneor more other components of the system 301 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 315 can store files, such as drivers, libraries andsaved programs. The storage unit 315 can store user data, e.g., userpreferences and user programs. The computer system 301 in some cases caninclude one or more additional data storage units that are external tothe computer system 301, such as located on a remote server that is incommunication with the computer system 301 through an intranet or theInternet.

The computer system 301 can communicate with one or more remote computersystems through the network 330. For instance, the computer system 301can communicate with a remote computer system of a user (e.g., aphysician, a nurse, a caretaker, a patient, or a subject). Examples ofremote computer systems include personal computers (e.g., portable PC),slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab),telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device,Blackberry®), or personal digital assistants. The user can access thecomputer system 301 via the network 330.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 301, such as, for example, on the memory310 or electronic storage unit 315. The machine executable or machinereadable code can be provided in the form of software. During use, thecode can be executed by the processor 305. In some cases, the code canbe retrieved from the storage unit 315 and stored on the memory 310 forready access by the processor 305. In some situations, the electronicstorage unit 315 can be precluded, and machine-executable instructionsare stored on memory 310.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 301, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 301 can include or be in communication with anelectronic display 335 that comprises a user interface (UI) 340 forproviding, for example, determined quantitative measures generated froma blood sample of a subject, statistical measures of deviation of thecounts, and determined tumor progression or tumor non-progression of thesubject. Examples of UI's include, without limitation, a graphical userinterface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 305. Thealgorithm can, for example, acquire pixel information of an array ofbiological, chemical, or physical entities; and detect the array ofbiological, chemical, or physical entities based at least in part on theacquired pixel information.

EXAMPLES Example 1: Array of Light-Sensing Devices with FluorescentlyLabeled Biomolecules Bound to a Functionalized Area of the Chip Surface(e.g., Immobilized SNAPs) and a Probe

FIG. 4 illustrates a top view of an array of light-sensing devices withfluorescently labeled biomolecules bound to a functionalized area of thechip surface (e.g., immobilized SNAPs) and a probe, in accordance withdisclosed embodiments. The array of light-sensing devices may beintegrated with digital logic (e.g., interface, timing, processing, andoutput). The array of light-sensing devices may be integrated with anintegrated image sensor (e.g., CMOS sensor) with functionalized areas.The CMOS sensor may be a CMOS active pixel sensor color imaging array.

FIG. 5 illustrates a cross-sectional view of one pixel of alight-sensing device with fluorescently labeled biomolecules bound to afunctionalized area of the chip surface (e.g., immobilized SNAPs) and aprobe, in accordance with disclosed embodiments. Incident light may becollected by an on-chip lens and then processed by a color filter. Whenthe incident light hits the silicon surface on the backside of thesilicon substrate, a photo-diode can collect the photons of the incidentlight and convert the photons into electrons. In some embodiments, anon-chip lens may not be needed.

Example 2: Array of Light-Sensing Devices with Fluorescently LabeledBiomolecules Bound to a Functionalized Area of the Chip Surface (e.g.,Immobilized SNAPs), a Probe, and a Differential Surface Coating

FIG. 6 illustrates a cross-sectional view of one pixel of alight-sensing device with fluorescently labeled biomolecules bound to afunctionalized area of the chip surface (e.g., immobilized SNAPs), aprobe, and a differential surface coating, in accordance with disclosedembodiments. A first surface coating or chemistry may bind to biologicalobjects to be immobilized. A second surface coating or chemistry mayprevent non-specific binding. Incident light may be collected by anon-chip lens and then processed by a color filter. When the incidentlight hits the silicon surface on the backside of the silicon substrate,a photo-diode can collect the photons of the incident light and convertthe photons into electrons. In some embodiments, an on-chip lens may notbe needed.

Example 3: Array of Light-Sensing Devices with Fluorescently LabeledBiomolecules Bound to a Functionalized Area of the Chip Surface (e.g.,Immobilized SNAPs), a Probe, and a Micro-Well to Prevent Cross TalkBetween Pixels

FIG. 7 illustrates a cross-sectional view of one pixel of alight-sensing device with fluorescently labeled biomolecules bound to afunctionalized area of the chip surface (e.g., immobilized SNAPs), aprobe, and a micro-well to prevent cross talk between pixels, inaccordance with disclosed embodiments. The microwell walls are made ofopaque and/or reflecting material. Incident light may be collected by anon-chip lens and then processed by a color filter. When the incidentlight hits the silicon surface on the backside of the silicon substrate,a photo-diode can collect the photons of the incident light and convertthe photons into electrons. In some embodiments, an on-chip lens may notbe needed.

Example 4: Array of Light-Sensing Devices with Fluorescently LabeledBiomolecules Bound to a Functionalized Area of the Chip Surface (e.g.,Immobilized SNAPs), a Probe, and a Micro-Well to Increase Collection andConversion of Emitted Light

FIG. 8 illustrates a cross-sectional view of one pixel of alight-sensing device with fluorescently labeled biomolecules bound to afunctionalized area of the chip surface (e.g., immobilized SNAPs), aprobe, and a micro-well to increase collection and conversion of emittedlight, in accordance with disclosed embodiments. The microwell wallscomprise photon-to-electron conversions layers that function asphotodiodes, and electron collection layers that are made of an opaquematerial. Incident light may be collected by an on-chip lens and thenprocessed by a color filter. When the incident light hits the siliconsurface on the backside of the silicon substrate, a photo-diode cancollect the photons of the incident light and convert the photons intoelectrons. In addition, the microwell walls also collect and convertemitted light. In some embodiments, an on-chip lens may not be needed.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1.-27. (canceled)
 28. A method, comprising: (a) providing an array ofbiological entities on a substrate; (b) contacting the array ofbiological entities with a fluorescent detection reagent; wherein thefluorescent detection reagent comprises an affinity reagent attached toa first structured nucleic acid particle, and wherein the firststructured nucleic acid particle comprises a nucleic acid origami; and(c) detecting presence or absence of a fluorescent signal from thefluorescent detection reagent at each biological entity of the array ofbiological entities.
 29. The method of claim 28, wherein a biologicalentity of the array of biological entities is immobilized to thesubstrate by a second structured nucleic acid particle.
 30. The methodof claim 29, wherein providing the array of biological entities on thesubstrate comprises immobilizing the biological entities on thesubstrate.
 31. The method of claim 30, wherein immobilizing thebiological entity to the substrate comprises attaching the secondstructured nucleic acid particle to the substrate.
 32. The method ofclaim 30, further comprising, before immobilizing the array ofbiological entities on the substrate, attaching the biological entity ofthe array of biological entities to the second structured nucleic acidparticle.
 33. The method of claim 29, wherein the second structurednucleic acid particle comprises a short nucleic acid strand hybridizedto a long nucleic acid strand.
 34. The method of claim 33, wherein thesecond structured nucleic acid particle comprises a nucleic acidorigami.
 35. The method of claim 30, wherein immobilizing the array ofbiological entities on the substrate comprises non-covalently attachingthe array of biological entities on the substrate.
 36. The method ofclaim 30, wherein immobilizing the array of biological entities on thesubstrate comprises covalently attaching the array of biologicalentities on the substrate.
 37. The method of claim 29, wherein thesecond structured nucleic acid particle is coupled to the substrate by afunctional group.
 38. The method of claim 37, wherein the secondstructured nucleic acid particle is coupled to the substrate by abinding between a first oligonucleotide and a second oligonucleotide,wherein the first oligonucleotide is attached to the substrate and thesecond oligonucleotide comprises a strand of the structured nucleic acidparticle.
 39. The method of claim 28, further comprising, beforeproviding the array of biological entities on the substrate, modifyingthe substrate with functional groups that enhance attachment of thebiological entities to the substrate.
 40. The method of claim 28,wherein the substrate comprises a passivating layer.
 41. The method ofclaim 40, wherein the functional groups and the passivating layer arepatterned on the substrate.
 42. The method of claim 28, wherein abiological entity of the array of biological entities comprises aprotein.
 43. The method of claim 28, wherein a biological entity of thearray of biological entities comprises a small molecule, a DNA, an RNA,a glycoprotein, a metabolite, a carbohydrate, an enzyme, or an antibody.44. The method of claim 28, wherein a biological entity of the array ofbiological entities comprises a single protein bound to a single secondstructured nucleic acid particle.
 45. The method of claim 28, whereinthe fluorescent detection reagent comprises two or more affinityreagents attached to the first structured nucleic acid particle.